Methods relating to lung cancer

ABSTRACT

The methods and assays described herein relate to detection, diagnosis, and treatment of lung cancer, e.g., by detecting the level of expression of certain miRNAs described herein and/or by therapeutically increasing the level of those miRNAs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. § 121 of co-pendingU.S. application Ser. No. 16/828,053 filed Mar. 24, 2020, which is adivisional under 35 U.S.C. § 121 of co-pending U.S. application Ser. No.16/391,473 filed Apr. 23, 2019 issued as U.S. Pat. No. 10,632,140 onApr. 28, 2020, which is a continuation under 35 U.S.C. § 120 of U.S.application Ser. No. 15/877,447 filed Jan. 23, 2018 issued as U.S. Pat.No. 10,314,855 on Jun. 11, 2019, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62/449,223 filed Jan. 23,2017, the contents of which are incorporated herein by reference intheir entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.CA152751 and CA214182 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML file format and is hereby incorporatedby reference in its entirety. Said XML copy, created on May 1, 2023 isnamed 701586-088722-US_SL.xml and is 14,727 bytes in size.

BACKGROUND

World-wide, lung cancer is one of the most frequently-occurring types ofcancer and is a leading cause of cancer-related mortality. Over 1.8million new cases are diagnosed each year, while 1.56 million individualdie due to lung cancer annually. Lung cancer remains the leading causeof cancer-related death in the United States and the world due, in largepart, to the inability to detect the disease at its earliest and curablestage. Development of improved diagnostics and therapeutics is criticalto providing improved care for lung cancer patients.

SUMMARY

By analyzing the lungs of lung cancer patients, it was found thatcertain microRNAs (miRNAs) were found to be under-expressed in lungcancer and are demonstrated to act as tumor suppressors in non-canceroustissue. Accordingly, expression of these miRNAs is diagnostic of thepresence of lung cancer in a patient, and methods of increasing theexpression of these miRNAs can be therapeutic.

In one aspect of any of the embodiments, described herein is a method oftreating lung cancer in a subject in need thereof, the method comprisingadministering to the subject an agonist of at least 1 miRNA selectedfrom the group consisting of: miR-146a-5p, miR-324-5p, miR-223-3p,miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p to thesubject. In one aspect of any of the embodiments, described herein is amethod of treating lung cancer in a subject in need thereof, the methodcomprising administering to the subject an agonist of at least 1 miRNAselected from Table 10 and/or an inhibitor of at least 1 miRNA selectedfrom Table 11 to the subject. In some embodiments of any of the aspects,the administering step comprises the administration of a vectorcomprising a nucleic acid encoding the agonist and/or inhibitor.

In one aspect of any of the embodiments, described herein is a methodcomprising: detecting the level of expression of at least 1 miRNAselected from the group consisting of: miR-146a-5p, miR-324-5p,miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, andmiR-582-5p; in a sample obtained from a subject. In one aspect of any ofthe embodiments, described herein is a method comprising: obtaining asample from a subject; and detecting the level of expression of at least1 miRNAs selected from the group consisting of: miR-146a-5p, miR-324-5p,miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, andmiR-582-5p; in the sample.

In one aspect of any of the embodiments, described herein is a methodcomprising: detecting the level of expression of at least 1 miRNAselected from Table 10 and/or Table 11 in a sample obtained from asubject. In one aspect of any of the embodiments, described herein is amethod comprising: obtaining a sample from a subject; and detecting thelevel of expression of at least 1 miRNA selected from Table 10 and/orTable 11 in the sample.

In one aspect of any of the embodiments, described herein is an assayfor detecting lung cancer a subject, the assay comprising: subjecting atest sample of a subject to at least one analysis to determine the levelof expression of at least 1 miRNAs selected from the group consistingof: miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,miR-221-3p-miR-505-3p, and miR-582-5p; wherein an expression level ofthe at least 1 miRNA which decreased relative to a reference level,indicates the presence of lung cancer. In one aspect of any of theembodiments, described herein is an assay for detecting lung cancer asubject, the assay comprising: subjecting a test sample of a subject toat least one analysis to determine the level of expression of at least 1miRNA selected from Table 10 and/or Table 11; wherein an expressionlevel of the at least 1 miRNA of Table 10 which is decreased relative toa reference level or an expression level of the at least 1 miRNA ofTable 11 which is increased relative to a reference level, indicates thepresence of lung cancer.

In one aspect of any of the embodiments, described herein is a method oftreating lung cancer in a subject in need thereof, the method comprisingadministering to the subject an agonist of at least 1 miRNA selectedfrom Table 10 or an inhibitor of at least 1 miRNA selected from Table 11to the subject. In some embodiments of any of the aspects, the subjectis administered an agonist of at least 1 miRNA selected from the groupconsisting of: miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p. In some embodimentsof any of the aspects, the administering step comprises theadministration of a vector comprising a nucleic acid encoding theagonist and/or inhibitor.

In some embodiments of any of the aspects, the level of expression isdetected for at least two miRNAs selected from the group consisting of:miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,miR-221-3p-miR-505-3p, and miR-582-5p. In some embodiments of any of theaspects, the level of expression is detected for at least three miRNAsselected from the group consisting of: miR-146a-5p, miR-324-5p,miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, andmiR-582-5p. In some embodiments of any of the aspects, the level ofexpression is detected for at least four miRNAs selected from the groupconsisting of: miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p. In some embodimentsof any of the aspects, the level of expression is detected for at leastfive miRNAs selected from the group consisting of: miR-146a-5p,miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p,and miR-582-5p. In some embodiments of any of the aspects, the level ofexpression is detected for at least six miRNAs selected from the groupconsisting of: miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p. In some embodimentsof any of the aspects, the level of expression is detected for at leastseven miRNAs selected from the group consisting of: miR-146a-5p,miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p,and miR-582-5p. In some embodiments of any of the aspects, the level ofexpression is detected for miR-146a-5p, miR-324-5p, miR-223-3p,miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p. In someembodiments of any of the aspects, the level of expression is detectedfor at least miR-146a-5p, miR-324-5p, miR-223-3p, and miR-223-5p. Insome embodiments of any of the aspects, the level of expression isdetected for at least miR-146a-5p.

In some embodiments of any of the aspects, the subject is a mammal. Insome embodiments of any of the aspects, the subject is a human. In someembodiments of any of the aspects, the subject is a current or formersmoker. In some embodiments of any of the aspects, the sample is abronchial brushing or nose epithelial sample. In some embodiments of anyof the aspects, the subject is at risk of developing lung cancer.

In some embodiments of any of the aspects, the detecting step comprisessequencing of miRNAs in the sample. In some embodiments of any of theaspects, the method further comprises detecting the expression level ofone or more mRNAs. In some embodiments of any of the aspects, theexpression level of no more than 100 miRNAs and/or mRNAs is detected.

In some embodiments of any of the aspects, the method further comprisesadministering an agonist of at least 1 miRNA selected from Table 10 oran inhibitor of at least one miRNA selected from Table 11 to thesubject. In some embodiments of any of the aspects, the method furthercomprises administering an agonist of at least 1 miRNA selected from thegroup consisting of: miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the enrichment of known smoking-related miRNAs by GSEA. Aset of 23 miRNAs that are expressed at lower levels in bronchial airwaysamples from current smokers are significantly enriched among the miRNAsmost repressed among current smokers in the current dataset (q<0.001).The bar below the graph shows all 463 miRNAs ranked from most induced insmokers to most repressed (as shown in the distribution oft statisticsat the bottom), while the vertical black lines show the position, withinthis ranked list, of the 23 miRNAs described above to have decreasedexpression in the bronchial airway of smokers. The running enrichmentscore, which has a significantly negative minimum, indicates that the 23miRNAs are among the miRNA most repressed among current smokers in thecurrent small RNA sequencing dataset.

FIGS. 2A-2D depict miRNAs significantly differentially expressed inbronchial epithelium between patients with and without lung cancer. FIG.2A depicts expression of hsa-miR146a-5p (P=0.0008, q=0.125). FIG. 2Bdepicts expression of hsa-miR-324-5p (P=0.0007, q=0.125). FIG. 2Cdepicts expression of hsa-miR-223-3p (P=0.0007, q=0.125). FIG. 2Ddepicts expression of hsa-miR-223-5p (P=0.0016, q=0.184).

FIGS. 3A-3D demonstrate that miRNAs with cancer-associated expressionare negatively correlated with their predicted targets. The distributionof miRNA-mRNA correlations for each miRNA and its predicted targets isshown with a solid line. The null distribution of miRNA-mRNAcorrelations for each miRNA and all nontargets is shown with a dashedline. The difference between the two distributions was tested using theKolmogorov-Smirnov test.

FIG. 4 depicts a graph of significantly differentially expressedmicroRNAs between current and former smokers (q<0.01). Some of thesemicroRNAs have been previously associated with smoking status, such asmiR-218, miR-365, miR-30 and miR-99a.

DETAILED DESCRIPTION

As described herein, the inventors have discovered that the level ofcertain miRNAs is decreased in lung cancer as opposed to healthy lungtissue. Accordingly, the level of these miRNAs can be used to diagnosis,detect, and/or to treat lung cancer. Additionally, reversing thedecrease in the level of the miRNAs can be used to treat lung cancer.

In one aspect of any of the embodiments, described herein is a methodcomprising detecting the level of expression of at least 1 miRNAselected from the group consisting of: miR-146a-5p, miR-324-5p,miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, andmiR-582-5p in a sample obtained from a subject. In one aspect of any ofthe embodiments, described herein is an assay for detecting lung cancera subject, the assay comprising: subjecting a test sample obtained froma subject to at least one analysis to determine the level of expressionof at least 1 miRNAs selected from the group consisting of: miR-146a-5p,miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p,and miR-582-5p; wherein an expression level of the at least 1 miRNAwhich decreased relative to a reference level, indicates the presence oflung cancer.

In some embodiments of any of the aspects, the methods and assaysdescribed herein relate to detecting the level of expression of at leasttwo miRNAs, at least three miRNAs, at least four miRNAs, at least fivemiRNAs, at least six miRNAs, at least seven miRNAs, or all of the miRNAsof the group consisting of miR-146a-5p, miR-324-5p, miR-223-3p,miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p. Where asubset of the 8 foregoing miRNAs is used, any combination of the miRNAscan be used in each of various embodiments of the aspects describedherein. For example, when the level of at least two miRNAs is detected,it is specifically contemplated herein that any pairwise combination ofthe ten miRNAs can be detected, e.g., any combination shown in Table 1.

TABLE 1 Contemplated exemplary combinations of miRNAs are indicated by“X” miR- miR-324- miR-223- miR-223- miR- miR-221- miR-505- miR-582-146a-5p 5p 3p 5p 450b-5p 3p 3p 5p miR-146a- X X X X X X X 5p miR-324- XX X X X X X 5p miR-223- X X X X X X X 3p miR-223- X X X X X X X 5pmiR-450b- X X X X X X X 5p miR-221- X X X X X X X 3p miR-505- X X X X XX X 3p miR-582- X X X X X X X 5p

In some embodiments of any of the aspects, the levels of expression isdetected for at least miR-146-5p, e.g., for miR-146-5p or miR-146-5p andat least one of the other miRNAs described herein. In some embodimentsof any of the aspects, the levels of expression is detected for at leastmiR-146-5p, miR-146a-5p, miR-324-5p, miR-223-3p, and miR-223-5p, e.g.,for those four miRNAs or those four miRNAs and at least one of thefurther miRNAs described herein.

As used herein, “miR-146a-5p” refers to a mature miRNA derived frommiR-146a. The sequences for the precursor and mature form are known fora variety of species, e.g. human miR-146a (NCBI Gene ID NO: 406938; NCBItranscript accession number NR 029701.1; SEQ ID NO: 1) and humanmiR-146a-5p (SEQ ID NO: 2). A “miR-146a-5p oligonucleotide” can be amiR-146a-5p (e.g., SEQ ID NO: 2) or a sequence encoding such anoligonucleotide, e.g. SEQ ID NO: 1. In some embodiments, miR-146a-5p canbe human miR-146a-5p, e.g., hsa-miR-146a-5p.

As used herein, “miR-324-5p” refers to a mature miRNA derived frommiR-324. The sequences for the precursor and mature form are known for avariety of species, e.g. human miR-324 (NCBI Gene ID NO: 442898; NCBItranscript accession number NR_029896.1; SEQ ID NO: 3) and humanmiR-324-5p (SEQ ID NO: 4). A “miR-324-5p oligonucleotide” can be amiR-324-5p (e.g., SEQ ID NO: 4) or a sequence encoding such anoligonucleotide, e.g. SEQ ID NO: 3. In some embodiments, miR-324-5p canbe human miR-324-5p, e.g., hsa-miR-324-5p.

As used herein, “miR-223-3p” refers to a mature miRNA derived frommiR-223. The sequences for the precursor and mature form are known for avariety of species, e.g. human miR-223 (NCBI Gene ID NO: 407008; NCBItranscript accession number NR_029637.1; SEQ ID NO: 5) and humanmiR-223-3p (SEQ ID NO: 6). A “miR-223-3p oligonucleotide” can be amiR-223-3p (e.g., SEQ ID NO: 6) or a sequence encoding such anoligonucleotide, e.g. SEQ ID NO: 5. In some embodiments, miR-223-3p canbe human miR-223-3p, e.g., hsa-miR-223-3p.

As used herein, “miR-223-5p” refers to a mature miRNA derived frommiR-223. The sequences for the precursor and mature form are known for avariety of species, e.g. human miR-223 (NCBI Gene ID NO: 407008; NCBItranscript accession number NR_029637.1; SEQ ID NO: 5) and humanmiR-223-5p (SEQ ID NO: 7). A “miR-223-5p oligonucleotide” can be amiR-223-5p (e.g., SEQ ID NO: 7) or a sequence encoding such anoligonucleotide, e.g. SEQ ID NO: 5. In some embodiments, miR-223-5p canbe human miR-223-5p, e.g., hsa-miR-223-5p.

As used herein, “miR-450b-5p” refers to a mature miRNA derived frommiR-450b. The sequences for the precursor and mature form are known fora variety of species, e.g. human miR-450b (NCBI Gene ID NO: 100126302;NCBI transcript accession number NR_030587.1; SEQ ID NO: 8) and humanmiR-450b-5p (SEQ ID NO: 9). A “miR-450b-5p oligonucleotide” can be amiR-450b-5p (e.g., SEQ ID NO: 9) or a sequence encoding such anoligonucleotide, e.g. SEQ ID NO: 8. In some embodiments, miR-450b-5p canbe human miR-450b-5p, e.g., hsa-miR-450b-5p.

As used herein, “miR-221-3p” refers to a mature miRNA derived frommiR-221. The sequences for the precursor and mature form are known for avariety of species, e.g. human miR-221 (NCBI Gene ID NO: 407006; NCBItranscript accession number NR_029635.1; SEQ ID NO: 10) and humanmiR-221-3p (SEQ ID NO: 11). A “miR-221-3p oligonucleotide” can be amiR-221-3p (e.g., SEQ ID NO: 11) or a sequence encoding such anoligonucleotide, e.g. SEQ ID NO: 10. In some embodiments, miR-221-3p canbe human miR-221-3p, e.g., hsa-miR-221-3p.

As used herein, “miR-505-3p” refers to a mature miRNA derived frommiR-505. The sequences for the precursor and mature form are known for avariety of species, e.g. human miR-505 (NCBI Gene ID NO: 574508; NCBItranscript accession number NR_030230.1; SEQ ID NO: 12) and humanmiR-505-3p (SEQ ID NO: 13). A “miR-505-3p oligonucleotide” can be amiR-505-3p (e.g., SEQ ID NO: 13) or a sequence encoding such anoligonucleotide, e.g. SEQ ID NO: 12. In some embodiments, miR-505-3p canbe human miR-505-3p, e.g., hsa-miR-505-3p.

As used herein, “miR-582-5p” refers to a mature miRNA derived frommiR-582. The sequences for the precursor and mature form are known for avariety of species, e.g. human miR-582 (NCBI Gene ID NO: 693167; NCBItranscript accession number NR_030308.1; SEQ ID NO: 14) and humanmiR-582-5p (SEQ ID NO: 15). A “miR-582-5p oligonucleotide” can be amiR-582-5p (e.g., SEQ ID NO: 15) or a sequence encoding such anoligonucleotide, e.g. SEQ ID NO: 14. In some embodiments, miR-582-5p canbe human miR-582-5p, e.g., hsa-miR-582-5p.

In one aspect of any of the embodiments, described herein is a methodcomprising detecting the level of expression of at least 1 miRNAselected from Table 10 and/or at least 1 miRNA selected from Table 11 ina sample obtained from a subject. In one aspect of any of theembodiments, described herein is an assay for detecting lung cancer asubject, the assay comprising: subjecting a test sample obtained from asubject to at least one analysis to determine the level of expression ofat least 1 miRNA selected from Table 10 and/or at least 1 miRNA selectedfrom Table 11 wherein an expression level of the at least 1 miRNA fromTable 10 which is decreased relative to a reference level, and/or anexpression level of the at least 1 miRNA from Table 11 which isincreased relative to a reference level, indicates the presence of lungcancer.

In some embodiments of any of the aspects, the methods and assaysdescribed herein relate to detecting the level of expression of at leasttwo miRNAs, at least three miRNAs, at least four miRNAs, at least fivemiRNAs, at least six miRNAs, at least seven miRNAs, or more miRNAs ofTables 10 and/or 11. In some embodiments of any of the aspects, themethods and assays described herein relate to detecting the level ofexpression of at least one of the miRNAs of the group consisting ofmiR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,miR-221-3p-miR-505-3p, and miR-582-5p and at least one further miRNAselected from Tables 10 and/or 11. Any combination of the miRNAs can beused in each of various embodiments of the aspects described herein.

TABLE 10 miRNAs downregulated in lung cancer microRNA name miR-324-5pmiR-223-3p miR-146a-5p miR-223-5p miR-769-3p miR-618 miR-338-5p miR-210miR-450a-5p miR-30b-5p miR-450a-5p miR-34a-5p miR-652-3p miR-107 miR-887miR-147b miR-31-5p miR-29c-5p miR-324-3p miR-345-5p miR-450b-5pmiR-769-5p miR-326 miR-940 let-7g-5p miR-582-5p miR-31-3p miR-378imiR-505-3p miR-1249 miR-221-3p miR-1260b miR-3200-3p miR-425-3p miR-4791miR-1296 miR-4677-3p

TABLE 11 miRNAs upregulated in lung cancer microRNA name miR-375miR-24-1-5p miR-130b-5p miR-183-5p miR-203a

In some embodiments of any of the aspects, the detecting step cancomprise sequencing miRNAs present in the sample. In some embodiments ofany of the aspects, the detecting step can comprise hybridization ofmiRNAs in the sample with probes, e.g., miRNA-specific probes.

In some embodiments, measurement of the level of a target and/ordetection of the level or presence of a target, e.g. of an expressionproduct (a RNA expression product of one of the genes described herein)can comprise a transformation. As used herein, the term “transforming”or “transformation” refers to changing an object or a substance, e.g.,biological sample, nucleic acid or protein, into another substance. Thetransformation can be physical, biological or chemical. Exemplaryphysical transformation includes, but is not limited to, pre-treatmentof a biological sample, e.g., from whole blood to blood serum bydifferential centrifugation. A biological/chemical transformation caninvolve the action of at least one enzyme and/or a chemical reagent in areaction. For example, a DNA sample can be digested into fragments byone or more restriction enzymes, or an exogenous molecule can beattached to a fragmented DNA sample with a ligase. In some embodiments,a nucleic acid sample can undergo enzymatic replication, e.g., bypolymerase chain reaction (PCR).

Transformation, measurement, and/or detection of a target molecule, e.g.a miRNA and/or mRNA as described herein can comprise contacting a sampleobtained from a subject with a reagent (e.g. a detection reagent) whichis specific for the target, e.g., a target-specific reagent. In someembodiments, the target-specific reagent is detectably labeled. In someembodiments, the target-specific reagent is capable of generating adetectable signal. In some embodiments, the target-specific reagentgenerates a detectable signal when the target molecule is present. Insome embodiments of any of the aspects, the target-specific reagenthybridizes to the target, e.g., through base pairing interactions.

Methods to measure gene expression products are known to a skilledartisan. In some embodiments, the methods described herein can relate todetermining the level of messenger RNA (mRNA) or miRNA described herein.Such molecules can be isolated, derived, or amplified from a biologicalsample, such as a bronchial brushing sample. Techniques for thedetection of mRNA and/or miRNA expression is known by persons skilled inthe art, and can include but not limited to, PCR procedures, RT-PCR,quantitative RT-PCR Northern blot analysis, differential geneexpression, RNAse protection assay, microarray based analysis,next-generation sequencing; hybridization methods, etc.

In some embodiments, the level of an miRNA and/or mRNA can be measuredby a quantitative sequencing technology, e.g. a quantitativenext-generation sequence technology. Methods of sequencing a nucleicacid sequence are well known in the art. Briefly, a sample obtained froma subject can be contacted with one or more primers which specificallyhybridize to a single-strand nucleic acid sequence flanking the targetgene sequence and a complementary strand is synthesized. In somenext-generation technologies, an adaptor (double or single-stranded) isligated to nucleic acid molecules in the sample and synthesis proceedsfrom the adaptor or adaptor compatible primers. In some third-generationtechnologies, the sequence can be determined, e.g. by determining thelocation and pattern of the hybridization of probes, or measuring one ormore characteristics of a single molecule as it passes through a sensor(e.g. the modulation of an electrical field as a nucleic acid moleculepasses through a nanopore). Exemplary methods of sequencing include, butare not limited to, Sanger sequencing, dideoxy chain termination,high-throughput sequencing, next generation sequencing, 454 sequencing,SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrentsequencing, sequencing by hybridization, nanopore sequencing, Helioscopesequencing, single molecule real time sequencing, RNAP sequencing, andthe like. Methods and protocols for performing these sequencing methodsare known in the art, see, e.g. “Next Generation Genome Sequencing” Ed.Michal Janitz, Wiley-VCH; “High-Throughput Next Generation Sequencing”Eds. Kwon and Ricke, Humanna Press, 2011; and Sambrook et al., MolecularCloning: A Laboratory Manual (4 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (2012); which are incorporated byreference herein in their entireties.

The nucleic acid sequences of the miRNAs described herein have beenassigned miR BASE and NCBI accession numbers for different species suchas human, mouse and rat. Accordingly, a skilled artisan can design anappropriate primer or hybridization probe based on the known sequencefor determining the expression level of the indicated molecule.

In some embodiments of any of the aspects, the methods and assaysdescribed herein can further comprise detecting the expression level ofone or more mRNAs.

In some embodiments of any of the aspects, the methods and assaysdescribed herein comprise detecting the expression level of no more than100 miRNAs and/or mRNAs. In some embodiments of any of the aspects, themethods and assays described herein comprise detecting the expressionlevel of no more than 20 miRNAs and/or mRNAs. In some embodiments of anyof the aspects, the methods and assays described herein comprisedetecting the expression level of no more than 50 miRNAs and/or mRNAs.

Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from aparticular biological sample using any of a number of procedures, whichare well-known in the art, the particular isolation procedure chosenbeing appropriate for the particular biological sample. For example,freeze-thaw and alkaline lysis procedures can be useful for obtainingnucleic acid molecules from solid materials; heat and alkaline lysisprocedures can be useful for obtaining nucleic acid molecules fromurine; and proteinase K extraction can be used to obtain nucleic acidfrom blood (Roiff, A et al. PCR: Clinical Diagnostics and Research,Springer (1994)).

The term “sample” or “test sample” as used herein denotes a sample takenor isolated from a biological organism, e.g., a bronchial brushing,epithiel cell, blood, or plasma sample from a subject. Exemplarybiological samples include, but are not limited to, a biopsy, a tumorsample, biofluid sample; serum; plasma; urine; saliva; and/or tissuesample etc. The term also includes a mixture of the above-mentionedsamples. The term “test sample” also includes untreated or pretreated(or pre-processed) biological samples. In some embodiments, a testsample can comprise cells from a subject. In some embodiments, the testsample can be a biopsy, tumor sample, or lung or respiratory sample.

In some embodiments, the test sample can be an untreated test sample. Asused herein, the phrase “untreated test sample” refers to a test samplethat has not had any prior sample pre-treatment except for dilutionand/or suspension in a solution. Exemplary methods for treating a testsample include, but are not limited to, centrifugation, filtration,sonication, homogenization, heating, freezing and thawing, andcombinations thereof. In some embodiments, the test sample can be afrozen test sample, e.g., a frozen tissue. The frozen sample can bethawed before employing methods, assays and systems described herein.After thawing, a frozen sample can be centrifuged before being subjectedto methods, assays and systems described herein. In some embodiments,the test sample is a clarified test sample, for example, prepared bycentrifugation and collection of a supernatant comprising the clarifiedtest sample. In some embodiments, a test sample can be a pre-processedtest sample, for example, supernatant or filtrate resulting from atreatment selected from the group consisting of centrifugation,filtration, thawing, purification, and any combinations thereof. In someembodiments, the test sample can be treated with a chemical and/orbiological reagent. Chemical and/or biological reagents can be employedto protect and/or maintain the stability of the sample, includingbiomolecules (e.g., nucleic acid and protein) therein, duringprocessing. The skilled artisan is well aware of methods and processesappropriate for pre-processing of biological samples required fordetermination of the level miRNAs as described herein.

In some embodiments of any of the aspects, the sample is a sample ofepithelial cells from the respiratory tract. In some embodiments of anyof the aspects, the sample is a bronchial brushing or nasal epithelialsample. In some embodiments of any of the aspects, the sample is anaspirate sample.

The test sample can be obtained by removing a sample from a subject, butcan also be accomplished by using a previously isolated sample (e.g.isolated at a prior timepoint and isolated by the same or anotherperson). In some embodiments of any of the aspects, the methods andassays described herein can further comprise a step of obtaining asample from a subject.

In some embodiments of any of the aspects, the subject is a mammal. Insome embodiments of any of the aspects, the subject is a primate. Insome embodiments of any of the aspects, the subject is a human.

A level which is less than a reference level can be a level which isless by at least about 10%, at least about 20%, at least about 50%, atleast about 60%, at least about 80%, at least about 90%, or less thanthe reference level. In some embodiments, a level which is less than areference level can be a level which is statistically significantly lessthan the reference level.

A level which is greater than a reference level can be a level which isgreater by at least about 10%, at least about 20%, at least about 50%,at least about 100%, at least about 200%, at least about 300% or morethan the reference level. A level which is greater than a referencelevel can be a level which is greater by at least about 1 σ on astatistical comparison with normal subjects, preferentially at least 2σ. In some embodiments, a level which is greater than a reference levelcan be a level which is statistically significantly greater than thereference level.

In some embodiments, the reference can be a level of the target moleculein a population of subjects who do not have or are not diagnosed ashaving, and/or do not exhibit signs or symptoms of a cancer (e.g. lungcancer). In some embodiments, the reference can also be a level ofexpression of the target molecule in a control sample, a pooled sampleof control individuals or a numeric value or range of values based onthe same. In some embodiments, the reference can be the level of atarget molecule in a sample obtained from the same subject at an earlierpoint in time, e.g., the methods described herein can be used todetermine if a subject's sensitivity to a given therapy is changing overtime or if the subject is at greater risk of having or developing lungcancer as compared to in the past.

In some embodiments of any of the aspects, the subject is a current orformer cigarette smoker. In some embodiments of any of the aspects, thesubject is a subject at risk of developing lung cancer, e.g., a subjectwith a family history of lung cancer, a subject who is a current orformer cigarette smoker, a subject exposed to one or more risk factorsfor lung cancer, and/or a subject displaying one or more symptoms of orassociated with lung cancer.

In some embodiments of any of the aspects described herein, the methodsor assays can comprise a further step of administering a treatment forlung cancer to the subject, e.g., if the expression level of at leastone miRNA described herein (e.g., one of the miRNAs of Table 1 or 10) isdetermined to be decreased relative to a reference level. In someembodiments of any of the aspects described herein, the methods orassays can comprise a further step of administering a treatment for lungcancer to the subject, e.g., if the expression level of at least onemiRNA of Table 11 is determined to be increased relative to a referencelevel. In one aspect of any of the embodiments, described herein is amethod of treating lung cancer in a subject in need thereof, the methodcomprising subjecting a test sample obtained from a subject to at leastone analysis to determine the level of expression of at least 1 miRNAsselected from the group consisting of: miR-146a-5p, miR-324-5p,miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, andmiR-582-5p; and administering a treatment for lung cancer to the subjectif the expression level of the at least 1 miRNA is decreased relative toa reference level. In one aspect of any of the embodiments, describedherein is a method of treating lung cancer in a subject in need thereof,the method comprising subjecting a test sample obtained from a subjectto at least one analysis to determine the level of expression of atleast 1 miRNAs selected from the group consisting of: miR-146a-5p,miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p,and miR-582-5p; and administering a treatment for lung cancer to thesubject when the expression level of the at least 1 miRNA is decreasedrelative to a reference level.

In one aspect of any of the embodiments, described herein is a method oftreating lung cancer in a subject in need thereof, the method comprisingsubjecting a test sample obtained from a subject to at least oneanalysis to determine the level of expression of at least 1 miRNAs fromTable 10 and/or at least one miRNA from Table 11; and administering atreatment for lung cancer to the subject if the expression level of theat least 1 miRNA from Table 10 is decreased relative to a referencelevel and/or if the at least one miRNA from Table 11 is increasedrelative to a reference level. In one aspect of any of the embodiments,described herein is a method of treating lung cancer in a subject inneed thereof, the method comprising subjecting a test sample obtainedfrom a subject to at least one analysis to determine the level ofexpression of at least 1 miRNAs selected from Table 10 and/or at leastone miRNA selected from Table 11; and administering a treatment for lungcancer to the subject when the expression level of the at least 1 miRNAof Table 10 is decreased relative to a reference level and/or when theexpression level of the at least 1 miRNA of Table 11 is decreasedrelative to a reference level.

In one aspect of any of the embodiments, described herein is a method oftreating lung cancer in a subject in need thereof, the method comprisingadministering a treatment for lung cancer to a subject determined tohave an expression level of at least 1 miRNA selected from the groupconsisting of: miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p in a sample obtainedfrom the subject which is decreased relative to a reference level. Inone aspect of any of the embodiments, described herein is a method oftreating lung cancer in a subject in need thereof, the method comprisingadministering a treatment for lung cancer to a subject determined tohave an expression level of at least 1 miRNA selected from Table 10 in asample obtained from the subject which is decreased relative to areference level and/or an expression level of at least 1 miRNA selectedfrom Table 11 in a sample obtained from the subject which is increasedrelative to a reference level.

In some embodiments of any of the aspects, a treatment for lung cancercan comprise an agonist of at least 1 miRNAs selected from the groupconsisting of: miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p. In some embodimentsof any of the aspects, a treatment for lung cancer can comprise anagonist of at least 1 miRNA selected from Table 10. In some embodimentsof any of the aspects, a treatment for lung cancer can comprise aninhibitor of at least 1 miRNA selected from Table 11.

As used herein, an “agonist” of the expression of an miRNA, e.g., refersto any agent that increases the expression and/or level of the miRNA,e.g. increases the expression of the miRNA by at least 10%, at least20%, at least 30%, at least 50%, at least 100%, at least 200%, at least500% or more. In some embodiments, the agonist can be a miRNAoligonucleotide and/or a vector encoding a miRNA oligonucleotide. Insome embodiments of any of the aspects, an agonist of an miRNA can bethe sequence of a naturally-occurring variant of the miRNA or the geneencoding the miRNA, e.g., a sequence of the miRNA as known in miRBase orthe NCBI database for human or other species. In some embodiments of anyof the aspects, an agonist of a miRNA can be a miRNA selected from thegroup consisting of miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p or a nucleic acidencoding one of the foregoing miRNAs.

In some embodiments of any of the aspects, an agonist of a miRNA can bea miRNA or a nucleic acid encoding an miR that has at least 90%, atleast 92%, at least 95%, at least 98%, or at least 99% sequence identitywith the target miRNA. In some embodiments of any of the aspects, anagonist of an miRNA can be a miRNA or a nucleic acid encoding an miRthat has at least 90%, at least 92%, at least 95%, at least 98%, or atleast 99% sequence identity with the target miRNA and shares theactivity of the wild-type and/or naturally-occurring miRNA.

As used herein, the term “inhibitor” refers to an agent which candecrease the expression of an miRNA, e.g. by at least 10% or more, e.g.by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95%or more, or 98% or more. The efficacy of an inhibitor of one or moretargets, e.g. its ability to decrease the level target miRNA can bedetermined, e.g. by measuring the level of an expression product of thetarget and/or the activity of the target. Methods for measuring thelevel of a given mRNA are known to one of skill in the art, e.g. RT-PCRwith primers can be used to determine the level of RNA. In someembodiments, the inhibitor can be an inhibitory nucleic acid; anaptamer; an antibody reagent; an antibody; or a small molecule. In someembodiments, the inhibitor can be an inhibitory nucleic acid.

Inhibitors of the expression of a given miRNA can be an inhibitorynucleic acid. In some embodiments, the inhibitory nucleic acid is aninhibitory RNA (iRNA). Double-stranded RNA molecules (dsRNA) have beenshown to block gene expression in a highly conserved regulatorymechanism known as RNA interference (RNAi). The inhibitory nucleic acidsdescribed herein can include an RNA strand (the antisense strand) havinga region which is 30 nucleotides or less in length, i.e., 15-30nucleotides in length, generally 19-24 nucleotides in length, whichregion is substantially complementary to at least part the targeted mRNAtranscript. The use of these iRNAs enables the targeted degradation oftargeted mRNAs, resulting in decreased expression and/or activity of thetarget.

As used herein, the term “iRNA” refers to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.In one embodiment, an iRNA as described herein effects inhibition of theexpression and/or activity of a target. In certain embodiments,contacting a cell with the inhibitor (e.g. an iRNA) results in adecrease in the target RNA level in a cell by at least about 5%, about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 99%, up to and including 100% ofthe target mRNA level found in the cell without the presence of theiRNA.

In some embodiments, the iRNA can be a dsRNA. A dsRNA includes two RNAstrands that are sufficiently complementary to hybridize to form aduplex structure under conditions in which the dsRNA will be used. Onestrand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of the target.The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. Generally, the duplex structure is between 15 and 30inclusive, more generally between 18 and 25 inclusive, yet moregenerally between 19 and 24 inclusive, and most generally between 19 and21 base pairs in length, inclusive. Similarly, the region ofcomplementarity to the target sequence is between 15 and 30 inclusive,more generally between 18 and 25 inclusive, yet more generally between19 and 24 inclusive, and most generally between 19 and 21 nucleotides inlength, inclusive. In some embodiments, the dsRNA is between 15 and 20nucleotides in length, inclusive, and in other embodiments, the dsRNA isbetween 25 and 30 nucleotides in length, inclusive. As the ordinarilyskilled person will recognize, the targeted region of an RNA targetedfor cleavage will most often be part of a larger RNA molecule, often anmRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway). dsRNAs having duplexes as short as 9 base pairs can, undersome circumstances, mediate RNAi-directed RNA cleavage. Most often atarget will be at least 15 nucleotides in length, preferably 15-30nucleotides in length.

In some embodiments of any of the aspects, the subject is contacted withand/or administered at least one nucleic acid encoding exogenous and/orectopic miRNA and/or inhibitory nucleic acids, e.g., the nucleic acid istranscribed after the administering step to provide exogenous and/orectopic miRNA and/or inhibitory nucleic acids to the subject.

In one aspect of any of the embodiments, described herein is a method oftreating lung cancer in a subject in need thereof, the method comprisingadministering to the subject an agonist of at least 1 miRNA selectedfrom the group consisting of: miR-146a-5p, miR-324-5p, miR-223-3p,miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p. In oneaspect of any of the embodiments, described herein is a method oftreating lung cancer in a subject in need thereof, the method comprisingadministering to the subject an agonist of at least 1 miRNA selectedfrom Table 10 and/or at an inhibitor of at least 1 miRNA selected fromTable 11.

In one aspect of any of the embodiments, described herein is apharmaceutical composition comprising an agonist of at least 1 miRNAselected from the group consisting of miR-146a-5p, miR-324-5p,miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, andmiR-582-5p. In one aspect of any of the embodiments, described herein isa pharmaceutical composition comprising an agonist of at least 1 miRNAselected from Table 10 and/or an inhibitor of at least 1 miRNA selectedfrom Table 11. As used herein, the term “pharmaceutical composition”refers to the active agent in combination with a pharmaceuticallyacceptable carrier e.g. a carrier commonly used in the pharmaceuticalindustry. The phrase “pharmaceutically acceptable” is employed herein torefer to those compounds, materials, compositions, and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio. In someembodiments of any of the aspects, a pharmaceutically acceptable carriercan be a carrier other than water. In some embodiments of any of theaspects, a pharmaceutically acceptable carrier can be a cream, emulsion,gel, liposome, nanoparticle, and/or ointment. In some embodiments of anyof the aspects, a pharmaceutically acceptable carrier can be anartificial or engineered carrier, e.g., a carrier that the activeingredient would not be found to occur in in nature.

In some embodiments, the methods described herein relate to treating asubject having or diagnosed as having lung cancer (e.g., small cell lungcancer or non-small cell lung cancer. Subjects having lung cancer can beidentified by a physician using current methods of diagnosing lungcancer. Symptoms and/or complications of lung cancer which characterizethese conditions and aid in diagnosis are well known in the art andinclude but are not limited to, coughing, coughing blood, shortness ofbreath, chest pain, wheezing, hoarseness, difficulty breathing,unexplained weight loss, bone pain, and headaches. Tests that may aid ina diagnosis of, e.g. lung cancer include, but are not limited to,x-rays, CT scan, sputum cytology, or biopsies. A family history of lungcancer, or exposure to risk factors for lung cancer (e.g. radon orasbestos exposure) can also aid in determining if a subject is likely tohave lung cancer or in making a diagnosis of lung cancer.

As used herein, the term “cancer” relates generally to a class ofdiseases or conditions in which abnormal cells divide without controland can invade nearby tissues. Cancer cells can also spread to otherparts of the body through the blood and lymph systems. There are severalmain types of cancer. Carcinoma is a cancer that begins in the skin orin tissues that line or cover internal organs. Sarcoma is a cancer thatbegins in bone, cartilage, fat, muscle, blood vessels, or otherconnective or supportive tissue. Leukemia is a cancer that starts inblood-forming tissue such as the bone marrow, and causes large numbersof abnormal blood cells to be produced and enter the blood. Lymphoma andmultiple myeloma are cancers that begin in the cells of the immunesystem. Central nervous system cancers are cancers that begin in thetissues of the brain and spinal cord.

In some embodiments of any of the aspects, the cancer is a primarycancer. In some embodiments of any of the aspects, the cancer is amalignant cancer. As used herein, the term “malignant” refers to acancer in which a group of tumor cells display one or more ofuncontrolled growth (i.e., division beyond normal limits), invasion(i.e., intrusion on and destruction of adjacent tissues), and metastasis(i.e., spread to other locations in the body via lymph or blood). Asused herein, the term “metastasize” refers to the spread of cancer fromone part of the body to another. A tumor formed by cells that havespread is called a “metastatic tumor” or a “metastasis.” The metastatictumor contains cells that are like those in the original (primary)tumor. As used herein, the term “benign” or “non-malignant” refers totumors that may grow larger but do not spread to other parts of thebody. Benign tumors are self-limited and typically do not invade ormetastasize.

A “cancer cell” or “tumor cell” refers to an individual cell of acancerous growth or tissue. A tumor refers generally to a swelling orlesion formed by an abnormal growth of cells, which may be benign,pre-malignant, or malignant. Most cancer cells form tumors, but some,e.g., leukemia, do not necessarily form tumors. For those cancer cellsthat form tumors, the terms cancer (cell) and tumor (cell) are usedinterchangeably.

A subject that has a cancer or a tumor is a subject having objectivelymeasurable cancer cells present in the subject's body. Included in thisdefinition are malignant, actively proliferative cancers, as well aspotentially dormant tumors or micrometastases. Cancers which migratefrom their original location and seed other vital organs can eventuallylead to the death of the subject through the functional deterioration ofthe affected organs.

Treatments for lung cancer can include radiation therapy, stereotacticbody radiotherapy, surgery (e.g. resection, lobectomy, orpneumonectomy), chemotherapy, treatment with afatinib, bevacizumab,certinib, crizotinib, erlotinib, nivoluman, or ramucirumab.

In some embodiments, the methods described herein comprise administeringan effective amount of a composition described herein, e.g. an agonistof an miRNA described herein to a subject in order to alleviate asymptom of a lung cancer. As used herein, “alleviating a symptom” isameliorating any condition or symptom associated with the disease. Ascompared with an equivalent untreated control, such reduction is by atleast 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more asmeasured by any standard technique. A variety of means for administeringthe compositions described herein to subjects are known to those ofskill in the art. Such methods can include, but are not limited to oral,parenteral, intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), pulmonary, cutaneous, topical, injection, orintratumoral administration. Administration can be local or systemic.

The term “effective amount” as used herein refers to the amount of anagent needed to alleviate at least one or more symptom of the disease ordisorder, and relates to a sufficient amount of pharmacologicalcomposition to provide the desired effect. The term “therapeuticallyeffective amount” therefore refers to an amount of the agent that issufficient to provide a particular effect when administered to a typicalsubject. An effective amount as used herein, in various contexts, wouldalso include an amount sufficient to delay the development of a symptomof the disease, alter the course of a symptom disease (for example butnot limited to, slowing the progression of a symptom of the disease), orreverse a symptom of the disease. Thus, it is not generally practicableto specify an exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the agent, which achieves a half-maximal inhibition ofsymptoms) as determined in cell culture, or in an appropriate animalmodel. Levels in plasma can be measured, for example, by highperformance liquid chromatography. The effects of any particular dosagecan be monitored by a suitable bioassay, e.g., assay for tumor growthand/or size, among others. The dosage can be determined by a physicianand adjusted, as necessary, to suit observed effects of the treatment.

In some embodiments, the agonist or inhibitor of a miRNA can be providedor administered on a vector, e.g., a viral vector. In some embodiments,the agonist or inhibitor of a miRNA can be provided or administered as agene therapy.

In some embodiments, the technology described herein relates to apharmaceutical composition comprising an agonist or inhibitor of a miRNAas described herein, and optionally a pharmaceutically acceptablecarrier. In some embodiments, the active ingredients of thepharmaceutical composition comprise an agonist or an inhibitor of amiRNA as described herein. In some embodiments, the active ingredientsof the pharmaceutical composition consist essentially of an agonist oran inhibitor of a miRNA as described herein. In some embodiments, theactive ingredients of the pharmaceutical composition consist of anagonist or an inhibitor of a miRNA as described herein. Pharmaceuticallyacceptable carriers and diluents include saline, aqueous buffersolutions, solvents and/or dispersion media. The use of such carriersand diluents is well known in the art. Some non-limiting examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments, the carrier inhibits the degradation of the active agent.

In some embodiments, the pharmaceutical composition as described hereincan be a parenteral dose form. Since administration of parenteral dosageforms typically bypasses the patient's natural defenses againstcontaminants, parenteral dosage forms are preferably sterile or capableof being sterilized prior to administration to a patient. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions. In addition, controlled-release parenteraldosage forms can be prepared for administration of a patient, including,but not limited to, DURO S®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, withoutlimitation: sterile water; water for injection USP; saline solution;glucose solution; aqueous vehicles such as but not limited to, sodiumchloride injection, Ringer's injection, dextrose Injection, dextrose andsodium chloride injection, and lactated Ringer's injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and propylene glycol; and non-aqueous vehicles suchas, but not limited to, corn oil, cottonseed oil, peanut oil, sesameoil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compoundsthat alter or modify the solubility of a pharmaceutically acceptablesalt of an agonist and/or inhibitor of a miRNA as disclosed herein canalso be incorporated into the parenteral dosage forms of the disclosure,including conventional and controlled-release parenteral dosage forms.

Pharmaceutical compositions can also be formulated to be suitable fororal administration, for example as discrete dosage forms, such as, butnot limited to, tablets (including without limitation scored or coatedtablets), pills, caplets, capsules, chewable tablets, powder packets,cachets, troches, wafers, aerosol sprays, or liquids, such as but notlimited to, syrups, elixirs, solutions or suspensions in an aqueousliquid, a non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil emulsion. Such compositions contain a predetermined amountof the pharmaceutically acceptable salt of the disclosed compounds, andmay be prepared by methods of pharmacy well known to those skilled inthe art. See generally, Remington: The Science and Practice of Pharmacy,21st Ed., Lippincott, Williams, and Wilkins, Philadelphia Pa. (2005).

In some embodiments of any of the aspects, the compositions describedherein can be administered by inhalation, e.g., as a vapor or aerosolformulation or by nebulization. For use as aerosols, a compositiondescribed herein can be provided in solution or suspension may bepackaged in a pressurized aerosol container together with suitablepropellants, for example, hydrocarbon propellants like propane, butane,or isobutane with conventional adjuvants. A composition described hereincan also be administered in a non-pressurized form such as in anebulizer or atomizer. In some embodiments, a composition can also beadministered directly to the airways in the form of a dry powder, e.g.,by use with an inhaler. Aerosols for the delivery to the respiratorytract are known in the art. See for example, Adjei, A. and Garren, J.Pharm. Res., 1: 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J.Pharm., 114: 111-115 (1995); Gonda, I. “Aerosols for delivery oftherapeutic and diagnostic agents to the respiratory tract,” in CriticalReviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990); Andersonet al., Am. Rev. Respir. Dis., 140: 1317-1324 (1989)) and have potentialfor the systemic delivery of peptides and proteins as well (Patton andPlatz, Advanced Drug Delivery Reviews, 8:179-196 (1992)); Timsina et.al., Int. J. Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol.Market, 4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W.,Aerosol Sci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10(1989)); Rudt, S. and R. H. Muller, J. Controlled Release, 22: 263-272(1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858(1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. andPlatz, R., Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug.Del. Rev., 5: 107-132 (1990); Patton, J. S., et al., Controlled Release,28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology(1996); Niven, R. W., et al., Pharm. Res., 12(9); 1343-1349 (1995); andKobayashi, S., et al., Pharm. Res., 13(1): 80-83 (1996), contents of allof which are herein incorporated by reference in their entirety.

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient'sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug's onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug. In some embodiments, the an agonist and/or inhibitor of amiRNA described herein can be administered in a sustained releaseformulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

In some embodiments of any of the aspects, the an agonist and/orinhibitor of a miRNA described herein is administered as a monotherapy,e.g., another treatment for the lung cancer is not administered to thesubject.

In some embodiments of any of the aspects, the methods described hereincan further comprise administering a second agent and/or treatment tothe subject, e.g. as part of a combinatorial therapy. Non-limitingexamples of a second agent and/or treatment can include lung cancertreatments as described elsewhere herein, and/or radiation therapy,surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib,AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103;alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above.

In addition, the methods of treatment can further include the use ofradiation or radiation therapy. Further, the methods of treatment canfurther include the use of surgical treatments.

One of skill in the art can readily identify a chemotherapeutic agent ofuse (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, EdwardChu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles ofCancer Therapy, Chapter 85 in Harrison's Principles of InternalMedicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era ofMolecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 inAbeloff's Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

In certain embodiments, an effective dose of a composition comprising anagonist and/or inhibitor of a miRNA as described herein can beadministered to a patient once. In certain embodiments, an effectivedose of a composition can be administered to a patient repeatedly. Forsystemic administration, subjects can be administered a therapeuticamount of a composition, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg,2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg,30 mg/kg, 40 mg/kg, 50 mg/kg, or more.

In some embodiments, after an initial treatment regimen, the treatmentscan be administered on a less frequent basis. For example, aftertreatment biweekly for three months, treatment can be repeated once permonth, for six months or a year or longer. Treatment according to themethods described herein can reduce levels of a marker or symptom of acondition, e.g. lung cancer by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% or more.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing schedule canvary from once a week to daily depending on a number of clinicalfactors, such as the subject's sensitivity to the active agent. Thedesired dose or amount of activation can be administered at one time ordivided into subdoses, e.g., 2-4 subdoses and administered over a periodof time, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments, administration can bechronic, e.g., one or more doses and/or treatments daily over a periodof weeks or months. Examples of dosing and/or treatment schedules areadministration daily, twice daily, three times daily or four or moretimes daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month,2 months, 3 months, 4 months, 5 months, or 6 months, or more. Acomposition can be administered over a period of time, such as over a 5minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

The dosage ranges for the administration of an agent according to themethods described herein depend upon, for example, the form of theagent, its potency, and the extent to which symptoms, markers, orindicators of a condition described herein are desired to be reduced,for example the percentage reduction desired for tumor growth. Thedosage should not be so large as to cause adverse side effects.Generally, the dosage will vary with the age, condition, and sex of thepatient and can be determined by one of skill in the art. The dosage canalso be adjusted by the individual physician in the event of anycomplication.

The efficacy of an agent described herein in, e.g. the treatment of acondition described herein, or to induce a response as described herein(e.g. lung cancer) can be determined by the skilled clinician. However,a treatment is considered “effective treatment,” as the term is usedherein, if one or more of the signs or symptoms of a condition describedherein are altered in a beneficial manner, other clinically acceptedsymptoms are improved, or even ameliorated, or a desired response isinduced e.g., by at least 10% following treatment according to themethods described herein. Efficacy can be assessed, for example, bymeasuring a marker, indicator, symptom, and/or the incidence of acondition treated according to the methods described herein or any othermeasurable parameter appropriate, e.g. tumor size and/or growth rate.Efficacy can also be measured by a failure of an individual to worsen asassessed by hospitalization, or need for medical interventions (i.e.,progression of the disease is halted). Methods of measuring theseindicators are known to those of skill in the art and/or are describedherein. Treatment includes any treatment of a disease in an individualor an animal (some non-limiting examples include a human or an animal)and includes: (1) inhibiting the disease, e.g., preventing a worseningof symptoms (e.g. pain or inflammation); or (2) relieving the severityof the disease, e.g., causing regression of symptoms. An effectiveamount for the treatment of a disease means that amount which, whenadministered to a subject in need thereof, is sufficient to result ineffective treatment as that term is defined herein, for that disease.Efficacy of an agent can be determined by assessing physical indicatorsof a condition or desired response. It is well within the ability of oneskilled in the art to monitor efficacy of administration and/ortreatment by measuring any one of such parameters, or any combination ofparameters. Efficacy can be assessed in animal models of a conditiondescribed herein, for example treatment of lung cancer in a mouse model.When using an experimental animal model, efficacy of treatment isevidenced when a statistically significant change in a marker isobserved, e.g. tumor size and/or growth rate.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment or agent) and can include, for example,a decrease by at least about 10%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, a “increase” is a statistically significant increasein such level.

The term “exogenous” refers to a substance present in a cell other thanits native source. The term “exogenous” when used herein can refer to anucleic acid (e.g., a miRNA or nucleic acid encoding a miRNA) that hasbeen introduced by a process involving the hand of man into a biologicalsystem such as a cell or organism in which it is not normally found andone wishes to introduce the nucleic acid or polypeptide into such a cellor organism. Alternatively, “ectopic” can refer to a nucleic acid thathas been introduced by a process involving the hand of man into abiological system such as a cell or organism in which it is found inrelatively low amounts and one wishes to increase the amount of thenucleic acid or polypeptide in the cell or organism, e.g., to createectopic expression or levels. In contrast, the term “endogenous” refersto a substance that is native to the biological system or cell.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomolgous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of, e.g.,lung cancer. A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. lung cancer) or one or more complications related to such acondition, and optionally, have already undergone treatment for thecondition or the one or more complications related to the condition.Alternatively, a subject can also be one who has not been previouslydiagnosed as having the condition or one or more complications relatedto the condition. For example, a subject can be one who exhibits one ormore risk factors for the condition or one or more complications relatedto the condition or a subject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA orcDNA. Suitable RNA can include, e.g., mRNA or miRNA.

In some embodiments of any of the aspects, a nucleic acid as describedherein (e.g. a miRNA or a nucleic acid encoding an miRNA) is comprisedby a vector. In some of the aspects described herein, a nucleic acidsequence as described herein, or any module thereof, is operably linkedto a vector. The term “vector”, as used herein, refers to a nucleic acidconstruct designed for delivery to a host cell or for transfer betweendifferent host cells. As used herein, a vector can be viral ornon-viral. The term “vector” encompasses any genetic element that iscapable of replication when associated with the proper control elementsand that can transfer gene sequences to cells. A vector can include, butis not limited to, a cloning vector, an expression vector, a plasmid,phage, transposon, cosmid, chromosome, virus, virion, etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g. 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the nucleic acid encoding a polypeptide as described hereinin place of non-essential viral genes. The vector and/or particle may beutilized for the purpose of transferring any nucleic acids into cellseither in vitro or in vivo. Numerous forms of viral vectors are known inthe art.

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, insome embodiments of any of the aspects, be combined with other suitablecompositions and therapies. In some embodiments of any of the aspects,the vector is episomal. The use of a suitable episomal vector provides ameans of maintaining the nucleotide of interest in the subject in highcopy number extra chromosomal DNA thereby eliminating potential effectsof chromosomal integration.

In some embodiments of any of the aspects, an agonist and/or inhibitorof an miRNA as described herein can comprise a modified nucleic acidsequence, e.g., it is chemically modified to enhance stability or otherbeneficial characteristics. The nucleic acids described herein may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Modificationsinclude, for example, (a) end modifications, e.g., 5′ end modifications(phosphorylation, conjugation, inverted linkages, etc.) 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with stabilizing bases,destabilizing bases, or bases that base pair with an expanded repertoireof partners, removal of bases (abasic nucleotides), or conjugated bases,(c) sugar modifications (e.g., at the 2′ position or 4′ position) orreplacement of the sugar, as well as (d) backbone modifications,including modification or replacement of the phosphodiester linkages.Specific examples of RNA compounds useful in the embodiments describedherein include, but are not limited to RNAs containing modifiedbackbones or no natural internucleoside linkages. RNAs having modifiedbackbones include, among others, those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified RNAs that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. In some embodiments of any of the aspects, themodified RNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones can include, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included. Modified RNAbackbones that do not include a phosphorus atom therein have backbonesthat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatoms and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages. These include those having morpholino linkages(formed in part from the sugar portion of a nucleoside); siloxanebackbones; sulfide, sulfoxide and sulfone backbones; formacetyl andthioformacetyl backbones; methylene formacetyl and thioformacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; others having mixed N, O, S andCH2 component parts, and oligonucleosides with heteroatom backbones, andin particular —CH2-NH—CH2-, —CH2-N(CH3)-O—CH2-[known as a methylene(methylimino) or MMI backbone], —CH2-O—N(CH3)-CH2-,—CH2-N(CH3)-N(CH3)-CH2- and —N(CH3)-CH2-CH2-[wherein the nativephosphodiester backbone is represented as —O—P—O—CH2-].

In other RNA mimetics suitable or contemplated for use as agonists orinhibitors, both the sugar and the internucleoside linkage, i.e., thebackbone, of the nucleotide units are replaced with novel groups. Thebase units are maintained for hybridization with an appropriate nucleicacid target compound. One such oligomeric compound, an RNA mimetic thathas been shown to have excellent hybridization properties, is referredto as a peptide nucleic acid (PNA). In PNA compounds, the sugar backboneof an RNA is replaced with an amide containing backbone, in particularan aminoethylglycine backbone. The nucleobases are retained and arebound directly or indirectly to aza nitrogen atoms of the amide portionof the backbone.

A RNA can also be modified to include one or more locked nucleic acids(LNA). A locked nucleic acid is a nucleotide having a modified ribosemoiety in which the ribose moiety comprises an extra bridge connectingthe 2′ and 4′ carbons. This structure effectively “locks” the ribose inthe 3′-endo structural conformation. The addition of locked nucleicacids has been shown to increase RNA stability in serum, and to reduceoff-target effects (Elmen, J. et al., (2005) Nucleic Acids Research33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843;Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

Modified RNAs can also contain one or more substituted sugar moieties.The RNAs, e.g., agonists and/or inhibitors, described herein can includeone of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-,S-, or N-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH2)nO]mCH3, O(CH2)·nOCH3, O(CH2)nNH2, O(CH2) nCH3,O(CH2)nONH2, and O(CH2)nON[((CH2)nCH3)]2, where n and m are from 1 toabout 10. In some embodiments of any of the aspects, dsRNAs include oneof the following at the 2′ position: C1 to C10 lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN,Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anRNA, or a group for improving the pharmacodynamic properties of an RNA,and other substituents having similar properties. In some embodiments ofany of the aspects, the modification includes a 2′ methoxyethoxy(2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH2)2ON(CH3)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′—OCH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA, particularly the 3′ position ofthe sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs andthe 5′ position of 5′ terminal nucleotide. RNAs may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

A nucleic acid as described herein can also include nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the nucleic acids featured in theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and areexemplary base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

The preparation of the modified nucleic acids, backbones, andnucleobases described above are well known in the art.

Another modification of a nucleic acid featured in the invention, e.g.,an agonist or inhibitor as described herein, involves chemically linkingto the nucleic acid to one or more ligands, moieties or conjugates thatenhance the activity, cellular distribution, pharmacokinetic properties,or cellular uptake of the RNA. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan etal., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. lung cancer. The term “treating” includes reducing oralleviating at least one adverse effect or symptom of a condition,disease or disorder associated with a condition. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. In some embodimentsof any of the aspects, a pharmaceutically acceptable carrier can be acarrier other than water. In some embodiments of any of the aspects, apharmaceutically acceptable carrier can be a cream, emulsion, gel,liposome, nanoparticle, and/or ointment. In some embodiments of any ofthe aspects, a pharmaceutically acceptable carrier can be an artificialor engineered carrier, e.g., a carrier that the active ingredient wouldnot be found to occur in in nature.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

One of skill in the art can readily identify a chemotherapeutic agent ofuse (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, EdwardChu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles ofCancer Therapy, Chapter 85 in Harrison's Principles of InternalMedicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era ofMolecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 inAbeloff's Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

In some embodiments of any of the aspects, the disclosure describedherein does not concern a process for cloning human beings, processesfor modifying the germ line genetic identity of human beings, uses ofhuman embryos for industrial or commercial purposes or processes formodifying the genetic identity of animals which are likely to cause themsuffering without any substantial medical benefit to man or animal, andalso animals resulting from such processes.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. These and other changes can be made to the disclosure inlight of the detailed description. All such modifications are intendedto be included within the scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method comprising:    -   detecting the level of expression of at least 1 miRNAs selected        from the group consisting of:        -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;            hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p,            hsa-miR-223-3p, hsa-miR-505-3p, and hsa-miR-582-5p;    -   in a sample obtained from a subject.-   2. The method of paragraph 1, wherein the level of expression is    detected for at least two miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.-   3. The method of paragraph 1, wherein the level of expression is    detected for at least three miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.-   4. The method of paragraph 1, wherein the level of expression is    detected for at least four miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.-   5. The method of paragraph 1, wherein the level of expression is    detected for at least five miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.-   6. The method of paragraph 1, wherein the level of expression is    detected for at least six miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.-   7. The method of paragraph 1, wherein the level of expression is    detected for at least seven miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.-   8. The method of paragraph 1, wherein the level of expression is    detected for at least eight miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.-   9. The method of paragraph 1, wherein the level of expression is    detected for at least nine miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.-   10. The method of paragraph 1, wherein the level of expression is    detected for miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;    hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,    hsa-miR-505-3p, and hsa-miR-582-5p.-   11. The method of any of paragraphs 1-10, wherein the level of    expression is detected for at least miR-146a-5p, miR-324-5p,    miR-223-3p, and miR-223-5p.-   12. The method of paragraphs 1-11, wherein the level of expression    is detected for at least miR-146a-5p.-   13. The method of any of paragraphs 1-12, wherein the subject is a    mammal.-   14. The method of paragraph 13, wherein the subject is a human.-   15. The method of any of paragraphs 1-14, wherein the subject is a    current or former smoker.-   16. The method of any of paragraphs 1-15, wherein the sample is a    bronchial brushing or nose epithelial sample.-   17. The method of any of paragraphs 1-16, wherein the subject is at    risk of developing lung cancer.-   18. The method of any of paragraphs 1-17, wherein the detecting step    comprises sequencing of miRNAs in the sample.-   19. The method of any of paragraphs 1-18, wherein the method further    comprises detecting the expression level of one or more mRNAs.-   20. The method of any of paragraphs 1-19, wherein the expression    level of no more than 100 miRNAs and/or mRNAs is detected.

The method of any of paragraphs 1-20, wherein, the method furthercomprises administering an agonist of at least 1 miRNAs selected fromthe group consisting of:

-   -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p    -   to the subject.

-   21. A method comprising:    -   obtaining a sample from a subject; and    -   detecting the level of expression of at least 1 miRNAs selected        from the group consisting of:        -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;            hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p,            hsa-miR-223-3p, hsa-miR-505-3p, and hsa-miR-582-5p;    -   in the sample.

-   22. An assay for detecting lung cancer a subject, the assay    comprising:    -   subjecting a test sample of a subject to at least one analysis        to determine the level of expression of    -   at least 1 miRNAs selected from the group consisting of:        -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;            hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p,            hsa-miR-223-3p, hsa-miR-505-3p, and hsa-miR-582-5p;    -   wherein an expression level of the at least 1 miRNA which        decreased relative to a reference level, indicates the presence        of lung cancer.

-   23. The assay of paragraph 22, wherein the level of expression is    detected for at least two miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.

-   24. The assay of paragraph 22, wherein the level of expression is    detected for at least three miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.

-   25. The assay of paragraph 22, wherein the level of expression is    detected for at least four miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.

-   26. The assay of paragraph 22, wherein the level of expression is    detected for at least five miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.

-   27. The assay of paragraph 22, wherein the level of expression is    detected for at least six miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.

-   28. The assay of paragraph 22, wherein the level of expression is    detected for at least seven miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.

-   29. The assay of paragraph 22, wherein the level of expression is    detected for at least eight miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.

-   30. The assay of paragraph 22, wherein the level of expression is    detected for at least nine miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p.

-   31. The assay of paragraph 22, wherein the level of expression is    detected for miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;    hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,    hsa-miR-505-3p, and hsa-miR-582-5p.

-   32. The assay of any of paragraphs 22-31, wherein the level of    expression is detected for at least miR-146a-5p, miR-324-5p,    miR-223-3p, and miR-223-5p.

-   33. The assay of any of paragraphs 22-32, wherein the level of    expression is detected for at least miR-146a-5p.

-   34. The assay of any of paragraphs 22-33, wherein the subject is a    mammal.

-   35. The assay of any of paragraphs 22-34, wherein the subject is a    human.

-   36. The assay of any of paragraphs 22-35, wherein the subject is a    current or former smoker.

-   37. The assay of any of paragraphs 22-36, wherein the sample is a    bronchial brushing or nose epithelial sample.

-   38. The assay of any of paragraphs 22-37, wherein the subject is at    risk of developing lung cancer.

-   39. The assay of any of paragraphs 22-38, wherein the detecting step    comprises sequencing of miRNAs in the sample.

-   40. The assay of any of paragraphs 22-39, wherein the method further    comprises detecting the expression level of one or more mRNAs.

-   41. The assay of any of paragraphs 22-40, wherein the expression    level of no more than 100 miRNAs and/or mRNAs is detected.

The method of any of paragraphs 1-20, wherein, the method furthercomprises administering an agonist of at least 1 miRNAs selected fromthe group consisting of:

-   -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;        hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,        hsa-miR-505-3p, and hsa-miR-582-5p to the subject.

-   42. A method of treating lung cancer in a subject in need thereof,    the method comprising    -   administering of the subject an agonist of at least 1 miRNAs        selected from the group consisting of:        -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;            hsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p,            hsa-miR-223-3p, hsa-miR-505-3p, and hsa-miR-582-5p.    -   to the subject.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method of treating lung cancer in a subject in need thereof,    the method comprising    -   administering to the subject an agonist of at least 1 miRNA        selected from the group consisting of:        -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;            miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p.    -   to the subject.-   2. A method of treating lung cancer in a subject in need thereof,    the method comprising administering to the subject an agonist of at    least 1 miRNA selected from Table 10 and/or an inhibitor of at least    1 miRNA selected from Table 11 to the subject.-   3. The method of any of paragraphs 1-2, wherein the administering    step comprises the administration of a vector comprising a nucleic    acid encoding the agonist and/or inhibitor.-   4. A method comprising:    -   detecting the level of expression of at least 1 miRNA selected        from the group consisting of:        -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;            miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p;    -   in a sample obtained from a subject.-   5. A method comprising:    -   obtaining a sample from a subject; and    -   detecting the level of expression of at least 1 miRNAs selected        from the group consisting of:        -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;            miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p;    -   in the sample.-   6. The method of paragraph 4 or 5, wherein the level of expression    is detected for at least two miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   7. The method of paragraph 4 or 5, wherein the level of expression    is detected for at least three miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   8. The method of paragraph 4 or 5, wherein the level of expression    is detected for at least four miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   9. The method of paragraph 4 or 5, wherein the level of expression    is detected for at least five miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   10. The method of paragraph 4 or 5, wherein the level of expression    is detected for at least six miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   11. The method of paragraph 4 or 5, wherein the level of expression    is detected for at least seven miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   12. The method of paragraph 4 or 5, wherein the level of expression    is detected for miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;    miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p.-   13. The method of any of paragraphs 4-12, wherein the level of    expression is detected for at least miR-146a-5p, miR-324-5p,    miR-223-3p, and miR-223-5p.-   14. The method of paragraphs 4-13, wherein the level of expression    is detected for at least miR-146a-5p.-   15. A method comprising:    -   detecting the level of expression of at least 1 miRNA selected        from Table 10 and/or Table 11 in a sample obtained from a        subject.-   16. A method comprising:    -   obtaining a sample from a subject; and    -   detecting the level of expression of at least 1 miRNA selected        from Table 10 and/or Table 11 in the sample.-   17. The method of any of paragraphs 1-16, wherein the subject is a    mammal.-   18. The method of paragraph 17, wherein the subject is a human.-   19. The method of any of paragraphs 1-18, wherein the subject is a    current or former smoker.-   20. The method of any of paragraphs 4-19, wherein the sample is a    bronchial brushing or nose epithelial sample.-   21. The method of any of paragraphs 4-20, wherein the subject is at    risk of developing lung cancer.-   22. The method of any of paragraphs 4-21, wherein the detecting step    comprises sequencing of miRNAs in the sample.-   23. The method of any of paragraphs 4-22, wherein the method further    comprises detecting the expression level of one or more mRNAs.-   24. The method of any of paragraphs 4-23, wherein the expression    level of no more than 100 miRNAs and/or mRNAs is detected.-   25. The method of any of paragraphs 4-24, wherein, the method    further comprises administering an agonist of at least 1 miRNA    selected from Table 10 or an inhibitor of at least one miRNA    selected from Table 11 to the subject.-   26. The method of any of paragraphs 44-25, wherein, the method    further comprises administering an agonist of at least 1 miRNA    selected from the group consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p    -   to the subject.-   27. An assay for detecting lung cancer a subject, the assay    comprising:    -   subjecting a test sample of a subject to at least one analysis        to determine the level of expression of    -   at least 1 miRNAs selected from the group consisting of:        -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;            miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p;    -   wherein an expression level of the at least 1 miRNA which        decreased relative to a reference level, indicates the presence        of lung cancer.-   28. The assay of paragraph 27, wherein the level of expression is    detected for at least two miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   29. The assay of paragraph 27, wherein the level of expression is    detected for at least three miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   30. The assay of paragraph 27, wherein the level of expression is    detected for at least four miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   31. The assay of paragraph 27, wherein the level of expression is    detected for at least five miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   32. The assay of paragraph 27, wherein the level of expression is    detected for at least six miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   33. The assay of paragraph 27, wherein the level of expression is    detected for at least seven miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   34. The assay of paragraph 27, wherein the level of expression is    detected miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;    miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p.-   35. The assay of any of paragraphs 27-34, wherein the level of    expression is detected for at least miR-146a-5p, miR-324-5p,    miR-223-3p, and miR-223-5p.-   36. The assay of any of paragraphs 27-35, wherein the level of    expression is detected for at least miR-146a-5p.-   37. An assay for detecting lung cancer a subject, the assay    comprising:    -   subjecting a test sample of a subject to at least one analysis        to determine the level of expression of at least 1 miRNA        selected from Table 10 and/or Table 11;    -   wherein an expression level of the at least 1 miRNA of Table 10        which is decreased relative to a reference level or an        expression level of the at least 1 miRNA of Table 11 which is        increased relative to a reference level, indicates the presence        of lung cancer.-   38. The assay of any of paragraphs 27-37, wherein the subject is a    mammal.-   39. The assay of any of paragraphs 27-38, wherein the subject is a    human.-   40. The assay of any of paragraphs 27-39, wherein the subject is a    current or former smoker.-   41. The assay of any of paragraphs 27-40, wherein the sample is a    bronchial brushing or nose epithelial sample.-   42. The assay of any of paragraphs 27-41, wherein the subject is at    risk of developing lung cancer.-   43. The assay of any of paragraphs 27-42, wherein the detecting step    comprises sequencing of miRNAs in the sample.-   44. The assay of any of paragraphs 27-43, wherein the method further    comprises detecting the expression level of one or more mRNAs.-   45. The assay of any of paragraphs 27-44, wherein the expression    level of no more than 100 miRNAs and/or mRNAs is detected.-   46. The assay of any of paragraphs 27-45, wherein the method further    comprises administering an agonist of at least 1 miRNA selected from    Table 10 and/or an inhibitor of at least 1 miRNA selected from Table    11 to the subject.-   47. The assay of any of paragraphs 27-46, wherein the method further    comprises administering an agonist of at least 1 miRNAs selected    from the group consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p    -   to the subject.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method of treating lung cancer in a subject in need thereof,    the method comprising administering to the subject an agonist of at    least 1 miRNA selected from Table 10 or an inhibitor of at least 1    miRNA selected from Table 11 to the subject.-   2. The method of paragraph 1, wherein the subject is administered an    agonist of at least 1 miRNA selected from the group consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   3. The method of paragraph 1, wherein the administering step    comprises the administration of a vector comprising a nucleic acid    encoding the agonist and/or inhibitor.-   4. The method of paragraph 1, wherein the subject is a human.-   5. The method of paragraph 1, wherein the subject is a current or    former smoker.-   6. A method of treating lung cancer in a subject in need thereof,    the method comprising    -   administering a treatment for lung cancer to a subject        determined to have a level of expression of at least 1 miRNAs        selected from the group consisting of:        -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;            miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p;    -   in a sample obtained from the subject which is decreased        relative to a reference level.-   7. The method of paragraph 6, further comprising the first steps of    obtaining a sample from a subject; and determining the level of    expression of the at least 1 miRNA; and administering a treatment    for lung cancer if the level of expression of the at least 1 miRNA    is decreased relative to a reference level.-   8. The method of paragraph 6, wherein the level of expression is    detected for at least two miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   9. The method of paragraph 6, wherein the level of expression is    detected for at least three miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   10. The method of paragraph 6, wherein the level of expression is    detected for at least four miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   11. The method of paragraph 6, wherein the level of expression is    detected for at least five miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   12. The method of paragraph 6, wherein the level of expression is    detected for at least six miRNAs selected from the group consisting    of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   13. The method of paragraph 6, wherein the level of expression is    detected for at least seven miRNAs selected from the group    consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p.-   14. The method of paragraph 6, wherein the level of expression is    detected for miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;    miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p.-   15. The method of paragraph 6, wherein the level of expression is    detected for at least miR-146a-5p, miR-324-5p, miR-223-3p, and    miR-223-5p.-   16. The method of paragraph 6, wherein the level of expression is    detected for at least miR-146a-5p.-   17. The method of paragraph 6, wherein the sample is a bronchial    brushing or nose epithelial sample.-   18. The method of paragraph 6, wherein the method further comprises    detecting the expression level of one or more mRNAs.-   19. The method of paragraph 6, wherein the expression level of no    more than 100 miRNAs and/or mRNAs is detected.-   20. The method of paragraph 6, wherein the treatment for lung cancer    comprises administering an agonist of at least 1 miRNA selected from    the group consisting of:    -   miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,        miR-221-3p-miR-505-3p, and miR-582-5p    -   to the subject.

EXAMPLES Example 1

Bronchial brushings were collected from current and former smokersundergoing bronchoscopy for suspect lung cancer across 28 medicalcenters as part of the AEGIS clinical trials. MicroRNA expression wasprofiled via small RNA sequencing for 347 patients for which geneexpression data was also available. Described hereins are alterations inmicroRNA expression in the cytologically normal mainstem bronchus ofsmokers with lung cancer. Importantly, performance of existing bronchialgene-expression biomarkers for lung cancer can be significantly improvedby incorporating the expression of miR-146a-5p.

The methods described herein relate to the diagnosis, prognosis,detection, and/or treatment of lung cancer detection using bronchialbrushings collected from current and former smokers undergoingbronchoscopy for suspected lung cancer.

In some embodiments, by addition of microRNA expression, we improve theperformance of Percepta, the first commercially available bronchial geneexpression biomarker for lung cancer detection. In some embodiments, themethods described herein can be used in combination with, orconcurrently with PERCEPTA and/or the methods described in InternationalPatent Publication WO2005/000098; which is incorporated by referenceherein in its entirety.

Described herein are 8 cancer-associated microRNAs which can be microRNAtherapeutic targets for lung cancer. These microRNAs are under-expressedin lung cancer and can act as dysregulated tumor suppressors. It iscontemplated herein that their targets can reveal new underlyingmechanisms of lung cancer development.

Demonstrated herein is the use of bronchial microRNA data for lungcancer detection. microRNA data provides additional independentinformation to gene expression, that can be used to improve lung cancerbiomarkers.

Bronchial miR-146a-5p expression adds independent information toexisting bronchial gene expression biomarker(s). This microRNA is knownto control immune function, hematopoiesis and different cancermechanisms.

The biomarkers described herein can be used in combination with targetedtherapy to monitor the evolution the airway field of injury.

In some embodiments, described herein are integrative biomarkers of geneand microRNA expression. The proposed approach can further be extendedby integrating multiple datatypes, such as gene and protein expression,epigenetic data, somatic mutations, copy number data, etc.

In some embodiments, described herein is a clinically relevant test forlung cancer detection using bronchial microRNA and gene expression fromcurrent and former smokers undergoing bronchoscopy for suspected lung.This test improves the performance of Percepta, the first commerciallyavailable bronchial gene expression biomarker for lung cancer detection.Percepta's AUC is significantly improved (p-value 0.025).

Bronchoscopy is a low risk clinical procedure, therefore the methodsdescribed herein are a less invasive test compared to biopsy or surgery.Additionally, the methods described herein are relatively cheap comparedto CT screening, biopsy or surgery and moving the test into the nosetissue will make the procedure even less invasive.

Gene expression alterations in normal appearing epithelial cells fromthe mainstem bronchus can be used as an early detection biomarker forlung cancer among current and former smokers with suspect disease. It iscontemplated herein that microRNA expression in bronchial epithelialcells is associated with the presence of lung cancer and that in someembodiments, integrating microRNA and gene expression could yield a morerobust classifier.

As demonstrated herein, there are alterations in microRNA expression inthe cytologically normal ainstem bronchus of smokers with lung cancer.In addition, we identified microRNAs which regulate some of thebronchial gene-expression changes previously associated with lungcancer. Importantly, we found that the performance of an existingbronchial gene-expression biomarker for lung cancer can be improved byincorporating microRNA expression.

Patients under suspicion of having lung cancer are increasingly beingscreened with CT, leading to an increase in the number of discoveredsolitary pulmonary nodules. Depending on the patient's history andpreferences, the risk of cancer and the risk of surgery, as well as thecharacteristics of the nodule, these lesions are either seriallymonitored with imaging or the patients undergo invasive evaluation. Tofacilitate this decision, we recently developed [1] and validated [2] agene expression based classifier that distinguishes between smokers withand without lung cancer using mRNA isolated from cytologically normalcells in the mainstem bronchus. The fact that some genes aredifferentially expressed by cancer status in the normal appearing airwaysupports the idea of an airway molecular field of injury spanning therespiratory tract [3]. We hypothesize that bronchial microRNA expressionchanges, in addition to mRNA changes, may also be associated with thepresence of lung cancer and that integrating microRNA with geneexpression information could yield a more robust classifier.

MicroRNAs are a class of small noncoding RNAs that repress theexpression of their targets by binding to 3′ UTR complementary strands.microRNAs have been shown to be associated with various cancers and havebeen previously associated with the airway molecular field of injury. Inaddition, microRNAs are more stable molecules and easier to measure indegraded tissues than mRNAs [4].

Here we extend and improve the mRNA-based biomarker [1], [2] through theexploration of cancer-related microRNAs differentially expressed in theairway wide field of injury. Moreover, we aim to explain some of themRNA expression changes seen in the airway by identifying the miRNAsregulating these cancer related changes.

REFERENCES

-   [1] Duncan H Whitney, Michael R Elashoff, Kate Porta-Smith, Adam C    Gower, Anil Vachani, J Scott Ferguson, Gerard A Silvestri, Jerome S    Brody, Marc E Lenburg and Avrum Spira. Derivation of a bronchial    genomic classifier for lung cancer in a prospective study of    patients undergoing diagnostic bronchoscopy. BMC Medical Genomics.    8:18. 2015.-   [2] Gerard A. Silvestri, Anil Vachani, Duncan Whitney, Michael    Elashoff, Kate Porta Smith, J. Scott Ferguson, Ed Parsons, Nandita    Mitra, Jerome Brody, Marc E. Lenburg, and Avrum Spira, for the AEGIS    Study Team, A Bronchial Genomic Classifier for the Diagnostic    Evaluation of Lung Cancer. The New England Journal of    Medicine. 2015. DOI: 10.1056/NEJMoa1504601.-   [3] Avrum Spira, Jennifer E Beane, Vishal Shah, Katrina Steiling,    Gang Liu, Frank Schembri, Sean Gilman, YvesMartine Dumas, Paul    Calner, Paola Sebastiani, Sriram Sridhar, John Beamis, Carla Lamb,    Timothy Anderson, Norman Gerry, Joseph Keane, Marc E Lenburg and    Jerome S Brody. Airway epithelial gene expression in the diagnostic    evaluation of smokers with suspect lung cancer. Nature Medicine.    13(3): 361-366. 2007.-   [4] Etheridge A. et al. Extracellular microRNA: a new source of    biomarkers. Mutat Res. 717(1-2). 2011.-   [5] Perdomo C. et al. MicroRNA 4423 is a primate-specific regulator    of airway epithelial cell differentiation and lung carcinogenesis.    PNAS. 110(47): 18946-51. 2013.-   [6] Frank Schembri, Sriram Sridhar, Catalina Perdomo, Adam M.    Gustafson, Xiaoling Zhang, Ayla Ergun, Jining Lu, Gang Liu, Xiaohui    Zhang, Jessica Bowers, Cyrus Vaziri, Kristen Ott, Kelly Sensinger,    James J. Collins, Jerome S. Brody, Robert Getts, Marc E. Lenburg,    Avrum Spira. MicroRNAs as modulators of smoking-induced gene    expression changes in human airway epithelium. PNAS. 106(7):    2319-2324. 2009.-   [7] Catuogno S. et al. miR-34c may protect lung cancer cells from    paclitaxel-induced apoptosis. Oncogene. 32(3). 2013.-   [8] Chou Y. T. et al. EGFR promotes lung tumorigenesis by activating    miR-7 through a Ras/ERK/Myc pathway that targets the Ets2    transcriptional repressor ERF. Cancer Res. 70(21):8822-31. 2010.-   [9] Krysan K. et al. PGE2 driven expression of c-Myc and    oncomiR-17-92 contributes to apoptosis resistance in NSCLC. Mol.    Cancer Res. 12(5):765-74. 2014.-   [10] Nian W. et al. miR-223 functions as a potent tumor suppressor    of the Lewis lung carcinoma cell line by targeting insulin-like    growth factor-1 receptor and cyclin-dependent kinase 2. Oncol. Lett.    6(2). 2013.-   [11] Shen J. et al. Plasma microRNAs as potential biomarkers for    non-small-cell lung cancer. Lab. Invest. 91(4):579-87. 2011.-   [12] Zheng D. et al. Plasma microRNAs as novel biomarkers for early    detection of lung cancer. Int. J. Clin. Exp. Pathol. 4(6):575-86.    2011.-   [13] Shinuk Kim, Taesung Park, Mark Kon. Cancer survival    classification using integrated data sets and intermediate    information. Artificial Intelligence in Medicine. 62(1). 23-31.    2014.-   [14] Brase J. C. et al. Serum microRNAs as non-invasive biomarkers    for cancer. Mol. Cancer. 9(306). 2010.-   [15] Berindan-Neagoe I. and Calin G. A. Molecular Pathways:    microRNAs. Cancer Cells. and Microenvironment. Clin. Cancer Res.    20(24):6247-53. 2014.-   [16] Jiang F. et al. MiR-125b promotes proliferation and migration    of type II endometrial carcinoma cells through targeting TP53INP1    tumor suppressor in vitro and in vivo. BMC Cancer. 11(425). 2011.-   [17] Mitchell P. S. et al. Circulating microRNAs as stable    blood-based markers for cancer detection. PNAS. 105(30):10513-10518.    2008.-   [18] Nygaard S. et al. Identification and analysis of miRNAs in    human breast cancer and teratoma samples using deep sequencing. BMC    Med. Genomics. 2(35). 2009.-   [19] Taylor D. D. and Gercel-Taylor C. MicroRNA signatures of    tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.    Gynecol. Oncol. 110(1):13-21. 2008.-   [20] Labbaye C. and Testa U., The emerging role of MIR-146A in the    control of hematopoiesis, immune function and cancer. Journal of    Hematology and Oncology. 5(13). 2012.

Methods:

Using bronchial brushings collected from current and former smokersundergoing bronchoscopy for suspected lung cancer across 28 medicalcenters as part of the AEGIS clinical trials, we profiled microRNAexpression via small RNA sequencing for 347 patients for which geneexpression data was also available (Silvestri et al., NEJM 2015) and(BMC Medical Genomics 2015). First, we selected cancer-associatedmicroRNAs by linear modeling. Then, we explored the correlations betweenexpression of these microRNAs and their predicted mRNA targets. Lastly,we tested whether cancer-associated microRNA features improved theestablished bronchial gene-expression classifier proposed in (Silvestriet al., NEJM 2015) and (Whitney et al., BMC Medical Genomics 2015).

We developed the classifier using the following approach. First, wecalculated the prediction scores of the gene expression classifierproposed in (Silvestri et al., NEJM 2015) and (Whitney et al., BMCMedical Genomics 2015) using the subset of samples with matched mRNA andmicroRNA data. The discovery set (138 samples) is a subset of thepatients used to train the gene expression classifier and the test set(203 samples) is a subset of the test cohorts from (Silvestri et al.,NEJM 2015). The patient scores were obtained using a logistic regressionmodel which aggregates information from 17 most representativecancer-associated genes, patient's age, and genomic correlates ofsmoking status, pack years, and gender [18]. Then, we integrated thesescores with the expression of a single microRNA. We built a logisticregression model with two variables, the microRNA expression (e.g.miR-146a-5p) and gene-expression classifier score. Next, we trained theweights of the logistic regression in the training set and tested theperformance of this integrated score in the test set. The logisticregression model was implemented using cv.glmnet( ) function from glmnetR package. By training the weights of the logistic regression in thediscovery set, we obtained the following values: β1=1.8480041,β2=4.3879703, β3=−0.3724577. To evaluate the new classifier wecalculated performance metrics including the area under the receiveroperating characteristic curve (AUC), the specificity, and the negativepredictive value at 90% sensitivity. To compare the ROC curves of geneexpression classifier and the improved predictor, we used the DeLongtest from the pROC R package.

Results: We found that expression profiles of 42 microRNAs wereassociated with cancer status in the training set (p<0.05). Then weinvestigated the gene targets of the top differentially expressedmicroRNAs (FDR<0.2), such as miR-146a-5p, miR-324-5p, miR-223-3p,miR-223-5p. The expression values of these microRNAs were significantlynegatively correlated with the expression of their predicted mRNAtargets, compared to all microRNAs in the dataset (p<0.05). Their genetargets were enriched mainly in cancer associated pathways.

Furthermore, six microRNAs target one or more genes previouslyestablished to have cancer-associated expression. These microRNAs are:hsamiR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,hsa-miR-505-3p, hsa-miR-582-5p. Their gene targets play roles inretinoic acid metabolism, cell cycle signaling and submucosal glandmarkers. Moreover, we find that addition of miR-146a-5p, one of the topdifferentially expressed microRNAs whose expression decreases in thebronchial epithelium of cancer patients, to the existing gene expressionbiomarker significantly improves the classifier's performance (AUC) inthe 203-test set (p-value 0.025). The proposed integrative miRNA-mRNAbiomarker performs with an AUC of 0.71 in the test set.

In this study we have established that there are alterations in microRNAexpression in the cytologically normal mainstem bronchus of smokers withlung cancer. Additionally, we have shown that microRNA data providesadditional information to gene expression data and thus can be used toimprove lung cancer biomarkers.

Top four differentially expressed microRNAs which are significantlyassociated with lung cancer (FDR<0.2) are the following: miR-146a-5p,miR-324-5p, miR-223-3p, miR-223-5p. This finding is novel, since thesemicroRNAs have not been previously associated with the airway field ofinjury.

Moreover, six differentially expressed microRNAs (p<0.05), such ashsa-miR-450b-5p, hsa-miR-221-3p, hsa-miR-223-5p, hsa-miR-223-3p,hsa-miR-505-3p, hsa-miR-582-5p, regulate bronchial gene-expressionchanges previously associated with lung cancer. This is the first timethese microRNAs are associated with mRNA expression changes in theairway field of injury.

Importantly, we found that the performance of an existing bronchialgene-expression biomarker for lung cancer, previously proposed andtested in (Silvestri et al., NEJM 2015) and (BMC Medical Genomics 2015),is significantly improved by incorporating miR-146a-5p expression(p=0.025). This finding is also novel since miR-146a-5p expression hasnot been previously shown to improve lung cancer detection

Example 2

Gene expression alterations in normal-appearing bronchial epithelialcells can serve as a lung cancer detection biomarker in smokers. Giventhat miRNAs regulate airway gene expression responses to smoking, it wasevaluated whether miRNA expression is also altered in the bronchialepithelium of smokers with lung cancer. Using epithelial brushings fromthe mainstem bronchus of patients undergoing bronchoscopy for suspectedlung cancer (as part of the AEGIS-1/2 clinical trials), miRNA expressionwas profiled via small-RNA sequencing from 347 current and formersmokers for which gene expression data were also available. Patientswere followed for one year post bronchoscopy until a final diagnosis oflung cancer (n=194) or benign disease (n=153) was made. Followingremoval of 6 low-quality samples, 138 patients (AEGIS-1) were used as adiscovery set to identify four miRNAs (miR-146a-5p, miR-324-5p,miR-223-3p, and miR-223-5p) that were downregulated in the bronchialairway of lung cancer patients (ANOVA P<0.002, FDR<0.2). The expressionof these miRNAs is significantly more negatively correlated with theexpression of their mRNA targets than with the expression of othernontarget genes (K-S P<0.05). Furthermore, these mRNA targets areenriched among genes whose expression is elevated in cancer patients(GSEA FDR<0.001). Finally, it was found that the addition of miR-146a-5pto an existing mRNA biomarker for lung cancer significantly improves itsperformance (AUC) in the 203 samples (AEGIS-1/2) serving an independenttest set (DeLong P<0.05). These findings indicate that there are miRNAswhose expression is altered in the cytologically normal bronchialepithelium of smokers with lung cancer, and that they can regulatecancer-associated gene expression differences.

Lung cancer remains the leading cause of cancer-related death in theUnited States and the world due, in large part, to the inability todetect the disease at its earliest and curable stage. Once a pulmonarylesion is identified, physicians must decide between CT surveillanceversus airway/lung biopsy. When biopsy is required, the approach caninclude bronchos-copy, transthoracic needle biopsy (TTNB), or surgicallung biopsy (SLB). The choice among these procedures is determined onthe basis of considerations such as lesion size and location, thepresence of adenopathy, the risk associated with the procedure, andlocal expertise. Although bronchoscopy is relatively safe (less than 1%of procedures complicated by pneumothorax; ref. 1), this procedure islimited by its sensitivity (from 34% to 88%), depending on the locationand size of the lesion (2). Even with newer bronchoscopic guidancetechniques, the sensitivity for the detection of lung cancer is below70% for peripheral lesions (3).

A nondiagnostic bronchoscopy in this setting leads to a clinical dilemmaas to which of these patients should undergo further invasive diagnostictesting (TTNB or SLB). To facilitate this clinical decision, a geneexpression—based classifier was developed and validated thatdistinguishes between smokers with and without lung cancer using mRNAisolated from cytologically normal cells in the mainstem bronchus (4,5). This biomarker can improve the diagnostic sensitivity ofbronchoscopy for lung cancer detection.

The ability to identify gene expression changes associated with cancerstatus in the normal appearing airway supports the idea of an airwaymolecular field of injury spanning the respiratory tract (6). In thiscurrent study, the field of injury concept was expanded to miRNAs.miRNAs are a class of small, noncoding RNAs that repress gene expressionand protein translation of their targets by complementary binding to the3′ UTR of RNA transcripts. In addition, compared with mRNAs, miRNAs arethought to be more stable molecules, making them more easily measured indegraded tissues (7). Previous studies have shown that smoking altersthe expression of miRNAs in the bronchial airway epithelium (8, 9). Itwas contemplated herein that similar to mRNA, there might also be miRNAexpression changes associated with the presence of lung cancer inbronchial epithelium from the mainstem bronchus that may play a role inregulating cancer-associated gene expression differences and thatintegrating miRNA with gene expression could improve lung cancerdetection.

Materials and Methods

Patient Selection

As previously described, over 1,000 current and former smokersundergoing bronchoscopy for suspected lung cancer were enrolled in theAirway Epithelial Gene Expression in the Diagnosis of Lung Cancer(AEGIS) trials, two independent, prospective, multicenter, observationalstudies (registered as NCT01309087 and NCT00746759; refs. 4, 5).Exclusion criteria for patients enrolled in AEGIS trials were age lessthan 21 years, no history of smoking (defined as having smoked<100cigarettes), and a concurrent cancer diagnosis or history of lungcancer. All study protocols were approved by the Institutional ReviewBoard at each medical center, and written informed consent was obtainedfrom all patients prior to enrollment. Patients were followedprospectively for up to one year post-bronchoscopy until a finaldiagnosis was obtained.

In this study, miRNA expression was profiled via small RNA sequencingfor 347 AEGIS patients. In choosing patients to include in our study, wewere limited by patients with a benign diagnosis and matched themapproximately 1:1 with patients diagnosed with lung cancer. Moreover, itwas attempted to balance the cases and controls for smoking status,cumulative smoke exposure (pack-years), gender, and age. For all of thesamples selected for small RNA sequencing, gene expression profiling ofthe large RNA fraction had been performed previously using AffymetrixHuman Gene 1.0 ST arrays (4, 5) and was available for data integration.

138 (—40%) samples were assigned from AEGIS-1 to be used as a discoveryset (Table 2); these samples were drawn exclusively from the trainingset previously used to develop the gene expression classifier (4, 5).The remaining 203 samples comprise a test set (Table 2) and consistexclusively of samples from the AEGIS-1 (n=133) and AEGIS-2 (n=70) testsets that were previously used to validate the gene expressionclassifier (5).

TABLE 2 Patient Demographics Discovery set Test set n = 138 n = 203Cancer status (n)^(a) Lung cancer 88 103 Benign disease 50 100 Gender(n) Females 62 84 Males 76 119 Age (SD; n) 59 (11; 138) 59 (10; 203)Smoking status (n) Current 46 88 Former 92 115 Cumulative smokeexposure - 36 (24; 137) 37 (29; 199) pack-yr. (SD; n) Race (n) White 109149 Black 24 46 Unknown 5 8 Lesion size (n)  <3 cm 52 71 ≥3 cm 58 91Infiltrate 15 31 Unknown 13 10 Histology (n) NSCLC 72 79 NSCLC stage I11 16 II 3 5 III 15 19 IV 29 26 Not specified 14 13 NSCLC subtypeAdenocarcinoma 31 34 Squamous 27 25 Large cell 2 4 Not specified 12 16SCLC 16 21 SCLC stage Limited 4 8 Extensive 8 12 Not specified 4 1Uncertain histology 0 3 Diagnosis of benign disease (n) Resolution orstability 11 26 Alternative diagnosis 39 74 Type of alternativediagnosis Sarcoidosis 9 17 Inflammation 3 2 Fibrosis 1 1 Infection 8 14Other 18 40 NOTE: n indicates number of patients with available clinicaldata. Abbreviations: NSCLC, non-small cell lung cancer; SCLC, small-celllung cancer. ^(a)P < 0.05.

High-Throughput Sequencing of Small RNA

On the basis of previous work on the effect of multiplexing on miRNAexpression quantitation (10), 347 samples were sequenced in threebatches by multiplexing 12 samples per lane on an Illumina HiSeq 2000™.A total of 200 ng of total RNA from each sample was used for librarypreparation. The TruSeq Small RNA Sample Prep Kit™ (Illumina) was usedfor the first batch, while the NEBNext Multiplex Small RNA Library PrepSet™ (Illumina) was used for the second and third batches. RNA adapterswere ligated to 3′ and 5′ ends of the RNA, and the adapter-ligated RNAwas reverse transcribed into single-stranded cDNA. The RNA 3′ adapterwas designed to target miRNAs and other small RNAs that have a 3′hydroxyl group resulting from enzymatic cleavage by Dicer or other RNAprocessing enzymes. The cDNA was then amplified by PCR, using a commonprimer and a primer containing one of 12 index sequences. Theintroduction of the six-base index tag at the PCR step allowedmultiplexed sequencing of different samples in a single lane of aflowcell. A 0.5% PhiX spikein was also added in all lanes for qualitycontrol. Each multiplexed library was hybridized to one lane of the four8-lane High-Output single-read flow cells on a cBot Cluster GenerationSystem™ (Illumina) using TruSeq Single-Read Cluster Kit™ (Illumina). Theclustered flowcell was loaded onto a HiSeq 2000 sequencer for amultiplexed sequencing run, which consists of a standard 36-cyclesequencing read with the addition of a 7-cycle index read.

miRNA Alignment and Quality Control

To estimate miRNA expression, a small RNA sequencing pipeline describedpreviously was used (10). Briefly, the 30 adapter sequence was trimmedusing the FASTX toolkit. Reads longer than 15 nt were aligned to hg19using Bowtie™ v0.12.7 (11) allowing up to one mismatch and alignment toup to 10 genomic locations. miRNA expression was quantified by countingthe number of reads aligning to mature miRNA loci (miRBase v20) usingBedtools™ v2.9.0 (12, 13). miRNA counts within each sample werenormalized to log 2 RPM values by adding a pseudocount of one to eachmiRNA, dividing by the total number of reads that aligned to all miRNAloci within that sample, multiplying by 1×10⁶, and then applying a log₂transformation (10). The log 2 RPM expression values follow a normaldistribution by an Anderson-Darling test (P=2.2×10-16; ref 14).

Next, the distribution of read lengths present in each sample wasexamined to ensure that the sequences observed were of the proper lengthfor miRNA. The read length distribution ought to follow a normaldistribution with a mean of 22 bases. samples whose distribution had anabundance of reads well below or above the mean of 22 bases (with lessthan one million reads aligned to 22 read length) were filtered out,indicating that the sample was not properly sequenced, the adapters wereimproperly trimmed, or the sample was of poor quality. Six such sampleswere removed, leaving 341 samples included in the downstream analysis.In addition, miRNA loci with a low number of aligned reads were removed(less than 20 on average). A total of 463 miRNA loci passed the filterand were included in the analysis. Finally, ComBat™ (15) was applied tonormalize the miRNA expression in the three different batches.Large-scale variability in miRNA expression was examined by principalcomponents analysis. No outlier samples were detected using the firsttwo principal components, and there were no apparent global differencesin miRNA expression between samples from AEGIS-1 and AEGIS-2 (data notshown).

Data Availability

Raw FASTQ files as well as the normalized miRNA expression data areavailable on Gene Expression Omnibus (GEO) under the GEO accessionnumber GSE93284. mRNA data from Whitney and colleagues and Silvestri andcolleagues was used (GSE66499; refs. 4, 5).

Differential Expression Analysis

To identify smoking-associated miRNAs, while correcting for covariates,an F test (anova R function; ref 16) was applied between a multiplelinear regression (lm R function), with miRNA expression as the responsevariable, and smoking status, age, gender, cancer status, and pack-yearsas independent variables, and another multiple linear regression thatdid not include the smoking status as an independent variable.

Similarly, to identify miRNAs with cancer-associated expression patternsin the discovery cohort, while correcting for covariates, an F test wasapplied between a multiple linear regression, with miRNA expression asthe response variable, and cancer status, age, gender, smoking status,and pack-years as independent variables, and another multiple linearregression that did not include the cancer status as an independentvariable.

The P values were adjusted for FDR using Benjamini-Hochberg FDR (17) andare denoted with q-value.

Identifying miRNA-mRNA Relationships

The correlations between the differentially expressed miRNAs and theirtargets as predicted in the TargetScan database (18) were analyzed. Theconserved targets as defined in TargetScan 5 and 6 (8mer 0.8; 7mer-m81.3; 7mer-1A 1.6) were included. The probability of conserved targeting(19) has the advantage of identifying targeting interactions that arenot only more likely to e effective but also those that are more likelyto be consequential. Correlation coefficients were calculated usingPearson product moment coefficient. For each miRNA, the resultingdistribution of correlation coefficients were compared with thedistribution of correlation coefficients between the miRNA and all thegenes that have not been predicted to be targeted by it in TargetScan,using the Kolmogorov-Smirnov test.

Next, it was tested whether the negatively correlated targets(correlation FDR<0.1) of each differentially expressed miRNA wereenriched among the genes whose expression is associated with cancerstatus by gene set enrichment analysis (GSEA; ref 20). For thisenrichment analysis, genes were ranked by the t statistic of a multiplelinear regression, with miRNA expression as the response variable, andcancer status, age, gender, smoking status, and pack-years asindependent variables.

Incorporating miRNA Expression into the mRNA Classifier

First, the prediction score of the mRNA classifier was calculated (4,5). Then, for each cancer-associated miRNA, the mRNA classifier scorewas integrated with the miRNA's expression using logistic regression(glmnet R package). The coefficients of the logistic regression,corresponding to the intercept (α₀=1.8480041), weight of the classifierscore (α₁=4.3879703), and weight of the miRNA's expression(α₂=−0.3724577), were determined in the discovery set, and theperformance of the fully specified model was evaluated in theindependent test set samples. Classification performance was assessedusing the area under the receiver operating characteristic curve (ROCAUC). The statistical significance of the AUC improvement was computedby DeLong test (21) from the pROC R package (22).

Results

Patient Population

miRNA expression was profiled via small RNA sequencing for 347 patients(194 cancer-positive and 153 cancer-negative subjects) participating inthe AEGIS-1 and AEGIS-2 trials. Of the 347 miRNA samples, 341 passed thesequencing quality control filter (10). The characteristics of thediscovery set (138 samples) and the test set (203 samples) are shown inTable 2. Except for cancer status, the other clinical variables are notsignificantly different between the training and test sets. Significantassociations were also found between cancer status and age and lesionsize in the discovery set and with pack-years and lesion size in thetest set (data not shown).

Identifying Smoking-Associated miRNAs in Airway Epithelium

Previous work has shown that cigarette smoke creates a molecular fieldof injury throughout the airway, and specifically that miRNA expressionis altered with tobacco smoke exposure (9, 23-28). The ability to detectmiRNAs with smoking status-associated expression was used as a positivecontrol for the quality of the miRNA expression data. A set of 28 miRNAswas previously identified as modulators of smoking-related geneexpression changes in airway epithelium (9), with most of them (n=23)being downregulated in current smokers compared with never smokers. Itwas found that the miRNAs previously identified as being repressed bysmoking were significantly enriched among the miRNAs that were mostdownregulated in current smokers from AEGIS (GSEA q<0.001), as shown inFIG. 1 .

In addition, using the data described herein, significantlydifferentially expressed miRNAs between current and former smokers wereidentified by linear regression. 135 smoking-associated miRNAs werefound by P<0.05 (Table 4). The top 30 differentially expressed miRNAs inthe discovery set (q<0.01) are shown in Table 4. Among these, there werefound miRNAs whose expression has been previously associated withsmoking, such as miR-218, miR-365, miR-30a, and miR-99a (9).

The relationship between bronchial miRNA expression and otherpotentially relevant clinical variables, such as gender, age, andpack-years were also analyzed (Tables 5-7). It was found that inaddition to smoking status, gender is also associated with miRNAexpression (85 differentially expressed miRNAs, P<0.05).

Cancer-Associated miRNA Alterations in the Bronchial Airway Epithelium

Using the discovery set (n=138), 42 miRNAs were identified that showeddifferential expression between patients with and without cancer bylinear regression at a liberal P value threshold of P<0.05 (Table 8). Ofthese, four miRNA isoforms showed evidence of differential expression atFDR<0.2 (P<0.002). These four are: miR-146a-5p, miR-324-5p, miR-223-3p,and miR-223-5p. The expression profiles of these four miRNAs are shownin FIGS. 2A-2D. Consistent with the potential for these miRNAs tofunction as tumor suppressors, it was find that the four differentiallyexpressed miRNA isoforms are downregulated in the bronchial airway ofpatients with lung cancer.

Cancer-Associated miRNAs as Potential Regulators of the Airway GeneExpression Alterations

miRNAs often lead to the degradation of the mRNAs to which they bind.Therefore, it was sought to determine whether the expression of thesemiRNAs was negatively correlated with the expression of their genetargets. It was found that the distribution of the correlationcoefficients of each cancer-associated miRNA and its predicted mRNAtargets (binding site predicted targets from TargetScan) issignificantly more negative than the distribution of correlationcoefficients for nontarget genes (P<10-9 for each miRNA; FIGS. 3A-3D).

It was next sought to assess whether bronchial miRNA expression couldadd to the performance of an mRNA biomarker for lung cancer which waspreviously identified (4). Using the training set samples, logisticregression was used to build five cancer prediction models: one modelcontained the mRNA biomarker score alone, the other four modelscontained the mRNA biomarker score in combination with one of the fourmiRNAs identified as having significant cancer-associated expression.

Next, the ROC curve AUC of the mRNA biomarker was compared alone withthe four miRNA-containing models using a test set (Table 2) comprised ofAEGIS1 and AEGIS-2 samples that are independent of the AEGIS-1 samplesused to identify the four miRNAs with cancer-associated expression andindependent of the samples used to develop the mRNA biomarker. It wasfound that adding miR-146a-5p to the mRNA biomarker significantlyimproved the AUC in the test set, from 0.66 to 0.71 (P=0.025). The AUCof biomarkers incorporating either miR-324-5p or either of the twoisoforms of miR-223 was not significantly different than the AUC of themRNA biomarker alone (P>0.25) in the test set. The performance metricsof each miRNA combined with the mRNA biomarker are provided in Table 9.

Discussion

Bronchial airway gene expression differences between patients with andwithout lung cancer can be used as a biomarker with clinical utility inthe setting of patients with inconclusive results following bronchoscopyfor suspect lung cancer (4-6). It was determined herein that miRNAexpression was also be altered in the normal-appearing epithelium of themainstem bronchus, whether these miRNA expression differences play arole in regulating the observed gene expression differences, and thatlung cancer-associated miRNAs have the potential to aid in the detectionof disease.

Described herein are four miRNA isoforms (miR-146a-5p, miR-324-5p,miR-223-3p, and miR-223-5p) that have altered expression in the airwayepithelium of patients with lung cancer. That all four miRNAs havedecreased expression in the bronchial airway of lung cancer patients isconsistent with prior studies that have found miRNAs withcancer-specific expression, mostly downregulated, in tumors comparedwith normal tissue (34).

Although miRNA expression differences have been well documented intumors, these results are the first to demonstrate altered expression ofnot just these cancer-related miRNAs, but any miRNA in the bronchialairway of lung cancer patients. The expression of mRNAs that arepredicted targets of these miRNAs is significantly negativelycorrelated, suggesting that the expression of downstream genes isinduced as a consequence of the cancer-dependent loss of miRNAexpression.

Each differentially expressed miRNA's ability to enhance the performanceof an mRNA-based lung cancer biomarker was assessed and it was foundthat miR-146a-5p significantly improves performance. One possibleexplanation for why miR-223-3p and miR-223-5p did not improve biomarkerperformance is that one of their targets (SNCA) is already a componentof the mRNA classifier; thus, miR-223 expression might be substantiallyredundant with SNCA expression levels. If this hypothesis is correct, itwould suggest that miR-146a adds to the biomarker's performance becausethe mRNA biomarker does not already capture miR-146a-related expressioninformation.

In this study, demonstrated for the first time is the presence of anmiRNA field of injury in the bronchial airway for lung cancer.

REFERENCES

-   1. Tukey M H, Wiener R S. Population-based estimates of    transbronchial lung biopsy utilization and complications. Respir Med    2012; 106:1559-65.-   2. Rivera M P, Mehta A C, Wahidi M M. Establishing the diagnosis of    lung cancer: Diagnosis and management of lung cancer, 3rd ed:    American College of Chest Physicians evidence-based clinical    practice guidelines. Chest 2013; 143:e142S-65S.-   3. Wang Memoli J S, Nietert P J, Silvestri G A. Meta-analysis of    guided bronchoscopy for the evaluation of the pulmonary nodule.    Chest 2012; 142:385-93.-   4. Whitney D H, Elashoff M R, Porta-Smith K, Gower A C, Vachani A,    Ferguson J S, et al. Derivation of a bronchial genomic classifier    for lung cancer in a prospective study of patients undergoing    diagnostic bronchoscopy. BMC Med Genomics 2015; 8:18.-   5. Silvestri G A, Vachani A, Whitney D, Elashoff M, Smith K P,    Ferguson J S, et al. A bronchial genomic classifier for the    diagnostic evaluation of lung cancer. N Engl J Med 2015; 373:243-51.-   6. Spira A, Beane J E, Shah V, Steiling K, Liu G, Schembri F, et al.    Airway epithelial gene expression in the diagnostic evaluation of    smokers with suspect lung cancer. Nat Med 2007; 13:361-6.-   7. Etheridge A, Lee I, Hood L, Galas D, Wang K. Extracellular    microRNA: a new source of biomarkers. Mutat Res 2011; 717:85-90.-   8. Perdomo C, Campbell J D, Gerrein J, Tellez C S, Garrison C B,    Walser T C, et al. MicroRNA 4423 is a primate-specific regulator of    airway epithelial cell differentiation and lung carcinogenesis. Proc    Natl Acad Sci USA 2013; 110:18946-51.-   9. Schembri F, Sridhar S, Perdomo C, Gustafsond A M, Zhangd X, Ergun    A, et al. MicroRNAs as modulators of smoking-induced gene expression    changes in human airway epithelium. Proc Natl Acad Sci USA 2009;    106:2319-24.-   10. Campbell J D, Liu G, Luo L, Xiao J, Gerrein J, Juan-Guardela B,    et al. Assessment of microRNA differential expression and detection    in multiplexed small RNA sequencing data. RNA 2015; 21:164-71.-   11. Langmead B, Trapnell C, Pop M, Salzberg S L. Ultrafast and    memory efficient alignment of short DNA sequences to the human    genome. Genome Biol 2009; 10:R25.-   12. Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res    2004; 32: D109-11.-   13. Quinlan A R, Hall I M. BEDTools: a flexible suite of utilities    for comparing genomic features. Bioinformatics 2010; 26:841-2.-   14. Thode H C. Testing for normality. New York, N.Y.: CRC Press;    2002.-   15. Johnson W E, Li C, Rabinovic A. Adjusting batch effects in    microarray expression data using empirical Bayes methods.    Biostatistics 2007; 8:118-27.-   16. Chambers J. Linear models. Pacific Grove, C A: Wadsworth &    Brooks/Cole; 1992.-   17. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a    practical and powerful approach to multiple testing. J R Stat Soc    Series B Methodol 1995; 57:289-300.-   18. Lewis B P, Burge C B, Bartel D P. Conserved seed pairing, often    flanked by adenosines, indicates that thousands of human genes are    microRNA targets. Cell 2005; 120:15-20.-   19. Friedman R C, Farh K K-H, Burge C B, Bartel D P. Most mammalian    mRNAs are conserved targets of microRNAs. Genome Res 2008;    19:92-105.-   20. Subramanian A, Tamayo P, Mootha V K, Mukherjee S, Ebert B L,    Gillette M A, et al. Gene set enrichment analysis: a knowledge-based    approach for interpreting genome-wide expression profiles. Proc Natl    Acad Sci USA 2005; 102:15545-50.-   21. DeLong E R, DeLong D M, Clarke-Pearson D L. Comparing the Areas    under two or more correlated receiver operating characteristic    curves: a nonparametric approach. Biometrics 1988; 44:837.-   22. Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, Sanchez J C,    et al. pROC:an open-source package for R and Sb to analyze and    compare ROC curves. BMC Bioinformatics 2011; 12:77.-   23. Powell C A, Klares S, O'Connor G, Brody J S. Loss of    heterozygosity in epithelial cells obtained by bronchial brushing:    clinical utility in lung cancer. Clin Cancer Res 1999; 5:2025-34.-   24. Wistuba I I, Lam S, Behrens C, Virmani A K, Fong K M, LeRiche J,    et al. Molecular damage in the bronchial epithelium of current and    former smokers. J Natl Cancer Inst 1997; 89:1366-73.-   25. Franklin W A, Gazdar A F, Haney J, Wistuba I I, La Rosa F G,    Kennedy T, et al. Widely dispersed p53 mutation in respiratory    epithelium. A novel mechanism for field carcinogenesis. J Clin    Invest 1997; 100:2133-7.-   26. Guo M, House M G, Hooker C, Han Y, Heath E, Gabrielson E, et al.    Promoter hypermethylation of resected bronchial margins: a field    defect of changes? Clin Cancer Res 2004; 10:5131-6.-   27. Miyazu Y M. Telomerase expression in noncancerous bronchial    epithelia is a possible marker of early development of lung cancer.    Cancer Res 2005; 65:9623-7.-   28. Spira A, Beane J, Shah V, Liu G, Schembri F, Yang X, et al.    Effects of cigarette smoke on the human airway epithelial cell    transcriptome. Proc Natl Acad Sci USA 2004; 101:10143-8.-   29. Chen G, Umelo I A, Lv S, Teugels E, Fostier K, Kronenberger P,    et al. miR146a inhibits cell growth, cell migration and induces    apoptosis in nonsmall cell lung cancer cells. PLoS One 2013;    8:e60317.-   30. Labbaye C, Testa U. The emerging role of MIR-146A in the control    of hematopoiesis, immune function and cancer. J Hematol Oncol 2012;    5:13.-   31. Li G, Liu Y, Su Z, Ren S, Zhu G, Tian Y, et al. MicroRNA-324-3p    regulates nasopharyngeal carcinoma radioresistance by directly    targeting WNT2B. Eur J Cancer 2013; 49:2596-607.-   32. Nian W, Ao X, Wu Y, Huang Y, Shao J, Wang Y, et al. miR-223    functions as a potent tumor suppressor of the Lewis lung carcinoma    cell line by targeting insulin-like growth factor-1 receptor and    cyclin-dependent kinase 2. Oncol Lett 2013; 6:359-66.-   33. Huang D W, Sherman B T, Lempicki R A. Systematic and integrative    analysis of large gene lists using DAVID bioinformatics resources.    Nat Protoc 2009; 4:44-57.-   34. Lu J, Getz G, Miska E A, Alvarez-Saavedra E, Lamb J, Peck D, et    al. MicroRNA expression profiles classify human cancers. Nature    2005; 435:834-8.-   35. Kumaraswamy E, Wendt K L, Augustine L A, Stecklein S R, Sibala E    C, Li D, et al. BRCA1 regulation of epidermal growth factor receptor    (EGFR) expression in human breast cancer cells involves    micro-RNA-146a and is critical for its tumor suppressor function.    Oncogene 2015; 34:4333-46.-   36. Bhaumik D, Scott G K, Schokrpur S, Patil C K, Orjalo A V, Rodier    F, et al. MicroRNAs miR-146a/b negatively modulate the    senescence-associated inflammatory mediators IL-6 and IL-8. Aging    2009; 1:402-11.-   37. Mao X, Kikani C K, Riojas R A, Langlais P, Wang L, Ramos F J, et    al. APPL1 binds to adiponectin receptors and mediates adiponectin    signalling and function. Nat Cell Biol 2006; 8:516-23.-   38. Buechler C, Wanninger J, Neumeier M. Adiponectin receptor    binding proteins—recent advances in elucidating adiponectin    signalling pathways. FEBS Lett 2010; 584:4280-6.-   39. Singh B, Reddy P G, Goberdhan A, Walsh C, Dao S, Ngai I, et al.    p53 regulates cell survival by inhibiting PIK3C A in squamous cell    carcinomas. Genes Dev 2002; 16:984-93.-   40. Gustafson A M, Soldi R, Anderlind C, Scholand M B, Qian J, Zhang    X, et al. Airway PI3K pathway activation is an early and reversible    event in lung cancer development. Sci Transl Med 2010; 2:26ra25.

TABLE 3 Patient demographics stratified by cancer status. Theassociation of cancer with gender, smoking status and nodule size werecomputing using a Fisher's exact test; the association of cancer withage and cumulative smoke exposure were computed using a Student'st-test; n indicates number of patients with clinical data available; SDindicates standard deviation. Test set Discovery set n = 203 n = 138Lung Lung Cancer Benign Cancer Benign n = 88 n = 50 p n = 103 n = 100 pGender Females 25 37 0.84 38 46 0.2 Males 63 13 65 54 Age (SD; n) 61 560.01 60 58 0.29 (10; 88) (13; 50) (9; 103) (12; 100) Smoking Current 3214 0.35 47 41 0.57 Former 56 36 56 59 Cumulative Smoke 38 33 0.2 40 320.05 Exposure - pack-yr. (SD; n) (22; 88) (27; 49) (28; 102) (30; 97)Race White 69 40 0.98 74 75 0.8 Black 15  9 26 20 Unknown  4  1  3  5Lesion <3 cm 30 22 4 · 10⁻⁴ 20 51 8 · 10⁻¹⁴ Size >=3 cm 47 11 73 18Infiltrate  4 11  6 25 Unknown  7  6  4  6

TABLE 4 Smoking associated microRNAs (p < 0.05) Direction in miRBase IDmicroRNA name p-value t-statistic current smokers MI0000767_MIMAT0000710hsa-miR-365a-3p 6.99E−14 8.384407 UP MI0000769_MIMAT0022834hsa-miR-365b-3p 7.00E−14 8.384203 UP MI0005416_MIMAT0004284hsa-miR-675-5p 7.43E−12 7.527167 UP MI0000289_MIMAT0000270hsa-miR-181a-3p 1.97E−11 7.344121 UP MI0014228_MIMAT0015066hsa-miR-3065-5p 2.92E−11 7.269376 UP MI0000294_MIMAT0000275hsa-miR-218-5p 5.99E−11 −7.13266 DOWN MI0000295_MIMAT0000275hsa-miR-218-5p 6.17E−11 −7.12678 DOWN MI0014228_MIMAT0015378hsa-miR-3065-3p 8.02E−11 7.076626 UP MI0000683_MIMAT0000257hsa-miR-181b-5p 9.07E−11 7.053075 UP MI0000270_MIMAT0000257hsa-miR-181b-5p 9.43E−11 7.04563 UP MI0003137_MIMAT0002819hsa-miR-193b-3p 3.94E−10 6.768818 UP MI0000783_MIMAT0000728 hsa-miR-3752.92E−07 5.408163 UP MI0000269_MIMAT0000256 hsa-miR-181a-5p 1.63E−065.022647 UP MI0000289_MIMAT0000256 hsa-miR-181a-5p 1.63E−06 5.022619 UPMI0003782_MIMAT0006789 hsa-miR-1468 1.09E−05 −4.57523 DOWNMI0000254_MIMAT0004550 hsa-miR-30c-2-3p 1.17E−05 −4.55862 DOWNMI0003610_MIMAT0003266 hsa-miR-598 2.26E−05 4.396201 UPMI0005543_MIMAT0004927 hsa-miR-708-3p 2.89E−05 4.334139 UPMI0005543_MIMAT0004926 hsa-miR-708-5p 6.18E−05 4.140067 UPMI0000089_MIMAT0000089 hsa-miR-31-5p 9.02E−05 4.040998 UPMI0000078_MIMAT0004495 hsa-miR-22-5p 9.43E−05 4.02934 UPMI0000088_MIMAT0000088 hsa-miR-30a-3p 0.000115 −3.97679 DOWNMI0000269_MIMAT0004558 hsa-miR-181 a-2-3p 0.000137 −3.93045 DOWNMI0000737_MIMAT0001620 hsa-miR-200a-5p 0.000146 3.912748 UPMI0000273_MIMAT0004560 hsa-miR-183-3p 0.000157 3.893655 UPMI0000098_MIMAT0000095 hsa-miR-96-5p 0.000289 3.724799 UPMI0000101_MIMAT0000097 hsa-miR-99a-5p 0.000291 −3.72306 DOWNMI0000273_MIMAT0000261 hsa-miR-183-5p 0.000306 3.7089 UPMI0003139_MIMAT0002821 hsa-miR-181d 0.000411 −3.62574 DOWNMI0000078_MIMAT0000077 hsa-miR-22-3p 0.000626 3.505042 UPMI0000737_MIMAT0000682 hsa-miR-200a-3p 0.000845 3.416487 UPMI0000746_MIMAT0004678 hsa-miR-99b-3p 0.000904 −3.39641 DOWNMI0003820_MIMAT0003884 hsa-miR-454-5p 0.000906 −3.39591 DOWNMI0003205_MIMAT0002888 hsa-miR-532-5p 0.000947 −3.38262 DOWNMI0005545_MIMAT0004929 hsa-miR-190b 0.000975 −3.37397 DOWNMI0016436_MIMAT0018204 hsa-miR-676-3p 0.001084 −3.34211 DOWNMI0000081_MIMAT0000080 hsa-miR-24-3p 0.001205 3.310141 UPMI0000064_MIMAT0000064 hsa-let-7c 0.001216 −3.30728 DOWNMI0000080_MIMAT0000080 hsa-miR-24-3p 0.001217 3.307088 UPMI0000470_MIMAT0004603 hsa-miR-125b-2-3p 0.001225 −3.30498 DOWNMI0014186_MIMAT0015032 hsa-miR-3158-3p 0.001507 −3.24164 DOWNMI0014187_MIMAT0015032 hsa-miR-3158-3p 0.001534 −3.23617 DOWNMI0000460_MIMAT0004600 hsa-miR-144-5p 0.002046 −3.14643 DOWNMI0005562_MIMAT0004951 hsa-miR-887 0.002052 3.145495 UPMI0016010_MIMAT0018000 hsa-miR-23c 0.002269 3.113702 UPMI0001733_MIMAT0001636 hsa-miR-452-3p 0.002611 3.068962 UPMI0000079_MIMAT0000078 hsa-miR-23a-3p 0.003471 2.976788 UPMI0000301_MIMAT0000281 hsa-miR-224-5p 0.003558 2.968621 UPMI0000439_MIMAT0000418 hsa-miR-23b-3p 0.003769 2.949658 UPMI0002470_MIMAT0002177 hsa-miR-486-5p 0.003934 −2.93552 DOWNMI0000805_MIMAT0004694 hsa-miR-342-5p 0.003953 −2.93395 DOWNMI0000077_MIMAT0004494 hsa-miR-21-3p 0.004101 2.921693 UPMI0003591_MIMAT0003249 hsa-miR-584-5p 0.00437 −2.90054 DOWNMI0000750_MIMAT0000082 hsa-miR-26a-5p 0.004811 −2.86833 DOWNMI0000083_MIMAT0000082 hsa-miR-26a-5p 0.004813 −2.86818 DOWNMI0006415_MIMAT0005929 hsa-miR-1275 0.004879 −2.86359 DOWNMI0003581_MIMAT0003239 hsa-miR-574-3p 0.005154 2.845097 UPMI0000441_MIMAT0004589 hsa-miR-30b-3p 0.005343 −2.83292 DOWNMI0000234_MIMAT0000222 hsa-miR-192-5p 0.005851 −2.80193 DOWNMI0000448_MIMAT0000425 hsa-miR-130a-3p 0.005886 −2.79989 DOWNMI0000082_MIMAT0000081 hsa-miR-25-3p 0.005893 −2.7995 DOWNMI0000077_MIMAT0000076 hsa-miR-21-5p 0.006117 2.786672 UPMI0001733_MIMAT0001635 hsa-miR-452-5p 0.006251 2.779215 UPMI0000272_MIMAT0000259 hsa-miR-182-5p 0.006668 2.756908 UPMI0001729_MIMAT0001631 hsa-miR-451a 0.00711 −2.73462 DOWNMI0000089_MIMAT0004504 hsa-miR-31-3p 0.007343 2.72339 UPMI0003589_MIMAT0003247 hsa-miR-582-5p 0.007882 2.69853 UPMI0000748_MIMAT0000691 hsa-miR-130b-3p 0.008389 2.676552 UPMI0000088_MIMAT0000087 hsa-miR-30a-5p 0.008469 −2.67318 DOWNMI0000736_MIMAT0004674 hsa-miR-30c-1-3p 0.008581 −2.66855 DOWNMI0000272_MIMAT0000260 hsa-miR-182-3p 0.008731 2.662408 UPMI0000470_MIMAT0000423 hsa-miR-125b-5p 0.009243 −2.64211 DOWNMI0000446_MIMAT0000423 hsa-miR-125b-5p 0.009518 −2.63162 DOWNMI0000471_MIMAT0000445 hsa-miR-126-3p 0.009523 −2.63144 DOWNMI0003589_MIMAT0004797 hsa-miR-582-3p 0.010005 2.613703 UPMI0000743_MIMAT0004677 hsa-miR-34c-3p 0.010512 −2.59587 DOWNMI0000080_MIMAT0000079 hsa-miR-24-1-5p 0.010513 2.595842 UPMI0003834_MIMAT0003887 hsa-miR-769-3p 0.011142 2.574744 UPMI0000271_MIMAT0000258 hsa-miR-181c-5p 0.011176 −2.57363 DOWNMI0000271_MIMAT0004559 hsa-miR-181c-3p 0.01136 −2.5677 DOWNMI0000469_MIMAT0004602 hsa-miR-125a-3p 0.011383 −2.56696 DOWNMI0017290_MIMAT0019731 hsa-miR-4662a-5p 0.011406 −2.56622 DOWNMI0003575_MIMAT0004794 hsa-miR-551b-5p 0.012532 −2.53171 DOWNMI0006406_MIMAT0005923 hsa-miR-1269a 0.013649 −2.50014 DOWNMI0000434_MIMAT0004585 hsa-let-7i-3p 0.014389 2.480456 UPMI0000456_MIMAT0004597 hsa-miR-140-3p 0.014819 −2.46945 DOWNMI0016789_MIMAT0018965 hsa-miR-4446-3p 0.016347 −2.43247 DOWNMI0000764_MIMAT0000707 hsa-miR-363-3p 0.016384 −2.43161 DOWNMI0015995_MIMAT0017982 hsa-miR-3605-3p 0.017059 −2.41627 DOWNMI0003186_MIMAT0004775 hsa-miR-502-3p 0.017377 −2.40923 DOWNMI0001519_MIMAT0001413 hsa-miR-20b-5p 0.018296 −2.38951 DOWNMI0003657_MIMAT0020924 hsa-miR-642a-3p 0.018614 −2.38289 DOWNMI0000812_MIMAT0004700 hsa-miR-331-5p 0.018661 2.381911 UPMI0002470_MIMAT0004762 hsa-miR-486-3p 0.019508 −2.3648 DOWNMI0003190_MIMAT0004776 hsa-miR-505-5p 0.021653 −2.32421 DOWNMI0000101_MIMAT0004511 hsa-miR-99a-3p 0.021731 −2.3228 DOWNMI0000288_MIMAT0000269 hsa-miR-212-3p 0.02358 2.290676 UPMI0000253_MIMAT0000243 hsa-miR-148a-3p 0.023882 2.285637 UPMI0000107_MIMAT0000100 hsa-miR-29b-3p 0.02423 2.279899 UPMI0003184_MIMAT0002871 hsa-miR-500a-3p 0.024263 −2.27936 DOWNMI0000105_MIMAT0000100 hsa-miR-29b-3p 0.024364 2.277713 UPMI0000732_MIMAT0000460 hsa-miR-194-5p 0.025292 −2.26283 DOWNMI0000267_MIMAT0000254 hsa-miR-10b-5p 0.025327 −2.26227 DOWNMI0000086_MIMAT0000085 hsa-miR-28-5p 0.026716 2.240894 UPMI0000791_MIMAT0000738 hsa-miR-383 0.027841 −2.22429 DOWNMI0000434_MIMAT0000415 hsa-let-7i-5p 0.029044 −2.20717 DOWNMI0000488_MIMAT0000460 hsa-miR-194-5p 0.029417 −2.20198 DOWNMI0005561_MIMAT0004949 hsa-miR-877-5p 0.030033 2.193535 UPMI0003639_MIMAT0004808 hsa-miR-625-3p 0.030715 2.184364 UPMI0000460_MIMAT0000436 hsa-miR-144-3p 0.032348 −2.16311 DOWNMI0000812_MIMAT0000760 hsa-miR-331-3p 0.033042 2.154355 UPMI0016413_MIMAT0018183 hsa-miR-3909 0.033468 2.149058 UPMI0000113_MIMAT0000103 hsa-miR-106a-5p 0.034881 −2.13191 DOWNMI0000288_MIMAT0022695 hsa-miR-212-5p 0.035107 2.129219 UPMI0003685_MIMAT0003339 hsa-miR-421 0.036102 2.11756 UPMI0000439_MIMAT0004587 hsa-miR-23b-5p 0.03665 2.111253 UPMI0006358_MIMAT0005886 hsa-miR-1297 0.036658 −2.11116 DOWNMI0000462_MIMAT0000438 hsa-miR-152 0.036967 2.107647 UPMI0006357_MIMAT0005885 hsa-miR-1295a 0.037339 −2.10344 DOWNMI0006394_MIMAT0005911 hsa-miR-1260a 0.038609 −2.08937 DOWNMI0005765_MIMAT0004984 hsa-miR-941 0.03994 2.075042 UPMI0005763_MIMAT0004984 hsa-miR-941 0.03994 2.075042 UPMI0003132_MIMAT0003161 hsa-miR-493-3p 0.040122 −2.07311 DOWNMI0000342_MIMAT0000318 hsa-miR-200b-3p 0.041066 2.063223 UPMI0005530_MIMAT0002881 hsa-miR-509-3p 0.041551 −2.05823 DOWNMI0005717_MIMAT0002881 hsa-miR-509-3p 0.041551 −2.05823 DOWNMI0003196_MIMAT0002881 hsa-miR-509-3p 0.041551 −2.05823 DOWNMI0016053_MIMAT0018073 hsa-miR-3653 0.042775 −2.04582 DOWNMI0000742_MIMAT0004676 hsa-miR-34b-3p 0.043272 −2.04088 DOWNMI0000290_MIMAT0000271 hsa-miR-214-3p 0.044466 −2.02918 DOWNMI0015825_MIMAT0016847 hsa-miR-378c 0.045614 2.018188 UPMI0000481_MIMAT0000454 hsa-miR-184 0.047471 2.000892 UPMI0000479_MIMAT0000451 hsa-miR-150-5p 0.048959 −1.98744 DOWNMI0000094_MIMAT0000092 hsa-miR-92a-3p 0.049239 −1.98495 DOWNMI0003195_MIMAT0002880 hsa-miR-508-3p 0.049502 −1.98262 DOWN

TABLE 5 Gender associated microRNAs (p < 0.05) Direction miRBase IDmicroRNA name p-value t-statistic in males MI0003591_MIMAT0003249hsa-miR-584-5p 8.71E−05 4.050113 UP MI0006406_MIMAT0005923 hsa-miR-1269a0.00038 −3.64802 DOWN MI0000261_MIMAT0000250 hsa-miR-139-5p 0.0004213.619431 UP MI0002470_MIMAT0002177 hsa-miR-486-5p 0.00051 3.564016 UPMI0002470_MIMAT0004762 hsa-miR-486-3p 0.000597 3.518597 UPMI0000764_MIMAT0000707 hsa-miR-363-3p 0.001246 3.299905 UPMI0000482_MIMAT0000455 hsa-miR-185-5p 0.001287 3.290067 UPMI0005767_MIMAT0004985 hsa-miR-942 0.00245 3.089317 UPMI0001729_MIMAT0001631 hsa-miR-451a 0.00249 3.084152 UPMI0000267_MIMAT0000254 hsa-miR-10b-5p 0.003311 2.992199 UPMI0017878_MIMAT0021044 hsa-miR-5010-3p 0.003669 2.958545 UPMI0005524_MIMAT0004902 hsa-miR-891a 0.003731 2.95302 UPMI0000487_MIMAT0004614 hsa-miR-193a-5p 0.004155 2.917373 UPMI0000737_MIMAT0001620 hsa-miR-200a-5p 0.004183 −2.91512 DOWNMI0003205_MIMAT0002888 hsa-miR-532-5p 0.00421 2.912991 UPMI0016005_MIMAT0017994 hsa-miR-3615 0.004315 2.904792 UPMI0000460_MIMAT0004600 hsa-miR-144-5p 0.0044 2.898319 UPMI0000762_MIMAT0000705 hsa-miR-362-5p 0.004953 2.858559 UPMI0003132_MIMAT0003161 hsa-miR-493-3p 0.005383 2.830367 UPMI0000471_MIMAT0000445 hsa-miR-126-3p 0.00618 2.783127 UPMI0000113_MIMAT0000103 hsa-miR-106a-5p 0.00626 2.778729 UPMI0000743_MIMAT0000686 hsa-miR-34c-5p 0.008081 −2.68977 DOWNMI0003184_MIMAT0002871 hsa-miR-500a-3p 0.008227 2.683471 UPMI0006359_MIMAT0005887 hsa-miR-1299 0.008432 2.674752 UPMI0000482_MIMAT0004611 hsa-miR-185-3p 0.008837 2.658095 UPMI0000742_MIMAT0000685 hsa-miR-34b-5p 0.008861 −2.65714 DOWNMI0003129_MIMAT0002809 hsa-miR-146b-5p 0.008881 2.656329 UPMI0000737_MIMAT0000682 hsa-miR-200a-3p 0.009483 −2.63292 DOWNMI0000082_MIMAT0000081 hsa-miR-25-3p 0.009953 2.615567 UPMI0003583_MIMAT0003241 hsa-miR-576-5p 0.011609 2.559776 UPMI0000734_MIMAT0004672 hsa-miR-106b-3p 0.01165 2.55849 UPMI0003132_MIMAT0002813 hsa-miR-493-5p 0.011967 2.548663 UPMI0000461_MIMAT0000437 hsa-miR-145-5p 0.012154 2.542988 UPMI0014234_MIMAT0015072 hsa-miR-320e 0.012943 2.519831 UPMI0000748_MIMAT0000691 hsa-miR-130b-3p 0.013366 2.507912 UPMI0000471_MIMAT0000444 hsa-miR-126-5p 0.013573 2.502228 UPMI0000459_MIMAT0000435 hsa-miR-143-3p 0.014702 2.472407 UPMI0000448_MIMAT0000425 hsa-miR-130a-3p 0.015185 2.460286 UPMI0000438_MIMAT0004586 hsa-miR-15b-3p 0.01567 2.448448 UPMI0000814_MIMAT0004701 hsa-miR-338-5p 0.015785 2.445691 UPMI0000342_MIMAT0000318 hsa-miR-200b-3p 0.01587 −2.44367 DOWNMI0001519_MIMAT0001413 hsa-miR-20b-5p 0.016517 2.428529 UPMI0000300_MIMAT0004570 hsa-miR-223-5p 0.017687 2.402462 UPMI0000094_MIMAT0000092 hsa-miR-92a-3p 0.017863 2.398675 UPMI0000461_MIMAT0004601 hsa-miR-145-3p 0.018111 2.393409 UPMI0000434_MIMAT0000415 hsa-let-7i-5p 0.01864 2.382346 UPMI0000093_MIMAT0000092 hsa-miR-92a-3p 0.019155 2.37185 UPMI0000816_MIMAT0004703 hsa-miR-335-3p 0.019155 2.371841 UPMI0000438_MIMAT0000417 hsa-miR-15b-5p 0.020899 2.338061 UPMI0003129_MIMAT0004766 hsa-miR-146b-3p 0.021041 2.335417 UPMI0000281_MIMAT0000232 hsa-miR-199a-3p 0.021208 2.332323 UPMI0000282_MIMAT0004563 hsa-miR-199b-3p 0.02133 2.330082 UPMI0000242_MIMAT0000232 hsa-miR-199a-3p 0.021332 2.330055 UPMI0000115_MIMAT0000069 hsa-miR-16-5p 0.021953 2.318821 UPMI0000115_MIMAT0004518 hsa-miR-16-2-3p 0.022134 2.315603 UPMI0000070_MIMAT0000069 hsa-miR-16-5p 0.022163 2.315092 UPMI0005565_MIMAT0004954 hsa-miR-543 0.023505 2.291934 UPMI0003772_MIMAT0010214 hsa-miR-151b 0.023652 2.289467 UPMI0000290_MIMAT0000271 hsa-miR-214-3p 0.025275 2.263094 UPMI0000778_MIMAT0000722 hsa-miR-370 0.025438 2.260537 UPMI0003185_MIMAT0002872 hsa-miR-501-5p 0.026337 2.246644 UPMI0003667_MIMAT0003322 hsa-miR-652-3p 0.028277 2.218005 UPMI0000472_MIMAT0000446 hsa-miR-127-3p 0.031579 2.172998 UPMI0000460_MIMAT0000436 hsa-miR-144-3p 0.031737 2.170951 UPMI0000265_MIMAT0000252 hsa-miR-7-5p 0.031786 2.170322 UPMI0000264_MIMAT0000252 hsa-miR-7-5p 0.031836 2.169671 UPMI0000263_MIMAT0000252 hsa-miR-7-5p 0.032048 2.166942 UPMI0000255_MIMAT0000245 hsa-miR-30d-5p 0.033298 −2.15116 DOWNMI0000749_MIMAT0000692 hsa-miR-30e-5p 0.033377 −2.15018 DOWNMI0000790_MIMAT0000737 hsa-miR-382-5p 0.033437 2.14944 UPMI0000085_MIMAT0000084 hsa-miR-27a-3p 0.033468 −2.14906 DOWNMI0000747_MIMAT0004679 hsa-miR-296-3p 0.034018 2.142316 UPMI0003186_MIMAT0004775 hsa-miR-502-3p 0.034607 2.135181 UPMI0003632_MIMAT0003287 hsa-miR-618 0.035611 2.123284 UPMI0000802_MIMAT0004692 hsa-miR-340-5p 0.036036 −2.11833 DOWNMI0000090_MIMAT0004505 hsa-miR-32-3p 0.037585 −2.10069 DOWNMI0000456_MIMAT0004597 hsa-miR-140-3p 0.038434 2.091286 UPMI0000457_MIMAT0004598 hsa-miR-141-5p 0.03844 −2.09122 DOWNMI0000813_MIMAT0000762 hsa-miR-324-3p 0.038692 2.08846 UPMI0000786_MIMAT0000731 hsa-miR-378a-5p 0.041886 2.054805 UPMI0015995_MIMAT0017982 hsa-miR-3605-3p 0.0445 2.028849 UPMI0000650_MIMAT0000617 hsa-miR-200c-3p 0.045047 −2.02359 DOWNMI0020364_MIMAT0023712 hsa-miR-6087 0.047024 2.005 UPMI0003127_MIMAT0002808 hsa-miR-511 0.049781 1.980159 UPMI0000458_MIMAT0000433 hsa-miR-142-5p 0.049834 1.979692 UP

TABLE 6 Age associated microRNAs (p < 0.05) Direction with miRBase IDmicroRNA name p-value t-statistic age increase MI0000102_MIMAT0000098hsa-miR-100-5p 0.004609 2.882752 UP MI0000681_MIMAT0000646hsa-miR-155-5p 0.008066 −2.69041 DOWN MI0000289_MIMAT0000256hsa-miR-181a-5p 0.009424 −2.63516 DOWN MI0000269_MIMAT0000256hsa-miR-181a-5p 0.009427 −2.63504 DOWN MI0000289_MIMAT0000270hsa-miR-181a-3p 0.009663 −2.62621 DOWN MI0005416_MIMAT0004284hsa-miR-675-5p 0.012408 −2.53539 DOWN MI0003195_MIMAT0002880hsa-miR-508-3p 0.014651 2.473715 UP MI0000086_MIMAT0000085 hsa-miR-28-5p0.016109 −2.43802 DOWN MI0000683_MIMAT0000257 hsa-miR-181b-5p 0.016941−2.41891 DOWN MI0000270_MIMAT0000257 hsa-miR-181b-5p 0.01724 −2.41225DOWN MI0003198_MIMAT0002883 hsa-miR-514a-3p 0.024898 2.269085 UPMI0003200_MIMAT0002883 hsa-miR-514a-3p 0.024898 2.269085 UPMI0003199_MIMAT0002883 hsa-miR-514a-3p 0.024898 2.269085 UPMI0005767_MIMAT0004985 hsa-miR-942 0.030666 −2.18502 DOWNMI0003760_MIMAT0004819 hsa-miR-671-3p 0.032443 −2.1619 DOWNMI0000542_MIMAT0000510 hsa-miR-320a 0.033491 −2.14878 DOWNMI0000441_MIMAT0000420 hsa-miR-30b-5p 0.034698 2.134098 UPMI0003601_MIMAT0004800 hsa-miR-550a-5p 0.038568 −2.08981 DOWNMI0003600_MIMAT0004800 hsa-miR-550a-5p 0.038664 −2.08876 DOWNMI0000476_MIMAT0000430 hsa-miR-138-5p 0.041213 −2.06171 DOWNMI0000458_MIMAT0000433 hsa-miR-142-5p 0.043524 −2.03838 DOWNMI0000269_MIMAT0004558 hsa-miR-181 a-2-3p 0.04365 −2.03714 DOWNMI0000764_MIMAT0000707 hsa-miR-363-3p 0.04401 −2.03361 DOWNMI0000481_MIMAT0000454 hsa-miR-184 0.045025 2.023794 UPMI0000115_MIMAT0004518 hsa-miR-16-2-3p 0.047756 −1.99829 DOWN

TABLE 7 Pack-years (PY) associated microRNAs (p < 0.05) Direction withmiRBase ID microRNA name p-value t-statistic PY increaseMI0000071_MIMAT0000071 hsa-miR-17-3p 0.011764 −2.55466 DOWNMI0003183_MIMAT0002870 hsa-miR-499a-5p 0.016027 −2.4397 DOWNMI0015997_MIMAT0017985 hsa-miR-3607-3p 0.019021 2.374323 UPMI0000300_MIMAT0004570 hsa-miR-223-5p 0.019299 2.368731 UPMI0000434_MIMAT0004585 hsa-let-7i-3p 0.020197 −2.35112 DOWNMI0005544_MIMAT0004928 hsa-miR-147b 0.030047 2.193163 UPMI0000088_MIMAT0000088 hsa-miR-30a-3p 0.030984 2.180624 UPMI0019308_MIMAT0022494 hsa-miR-5701 0.036403 2.113925 UPMI0019593_MIMAT0022494 hsa-miR-5701 0.036403 2.113925 UPMI0000068_MIMAT0000067 hsa-let-7f-5p 0.037702 2.099219 UPMI0000067_MIMAT0000067 hsa-let-7f-5p 0.039373 2.080928 UPMI0003780_MIMAT0005794 hsa-miR-1296 0.039909 −2.0752 DOWNMI0000101_MIMAT0004511 hsa-miR-99a-3p 0.040476 −2.06922 DOWNMI0000300_MIMAT0000280 hsa-miR-223-3p 0.04588 2.015523 UP

TABLE 8 Cancer associated microRNAs (p < 0.05) Direction miRBase IDmicroRNA name p-value t-statistic in cancer MI0000813_MIMAT0000761hsa-miR-324-5p 0.000649 −3.49412 DOWN MI0000300_MIMAT0000280hsa-miR-223-3p 0.000714 −3.46634 DOWN MI0000477_MIMAT0000449hsa-miR-146a-5p 0.000808 −3.4298 DOWN MI0000300_MIMAT0004570hsa-miR-223-5p 0.001586 −3.22597 DOWN MI0003834_MIMAT0003887hsa-miR-769-3p 0.002842 −3.04179 DOWN MI0003632_MIMAT0003287 hsa-miR-6180.003085 −3.01518 DOWN MI0000814_MIMAT0004701 hsa-miR-338-5p 0.005204−2.8418 DOWN MI0000286_MIMAT0000267 hsa-miR-210 0.007292 −2.72581 DOWNMI0001652_MIMAT0001545 hsa-miR-450a-5p 0.008086 −2.68953 DOWNMI0000441_MIMAT0000420 hsa-miR-30b-5p 0.008275 −2.68142 DOWNMI0003187_MIMAT0001545 hsa-miR-450a-5p 0.008425 −2.67505 DOWNMI0000268_MIMAT0000255 hsa-miR-34a-5p 0.009728 −2.62381 DOWNMI0003667_MIMAT0003322 hsa-miR-652-3p 0.013213 −2.51217 DOWNMI0000114_MIMAT0000104 hsa-miR-107 0.016073 −2.43885 DOWNMI0000783_MIMAT0000728 hsa-miR-375 0.018023 2.395261 UPMI0000080_MIMAT0000079 hsa-miR-24-1-5p 0.018845 2.378128 UPMI0005562_MIMAT0004951 hsa-miR-887 0.020166 −2.35195 DOWNMI0005544_MIMAT0004928 hsa-miR-147b 0.022438 −2.31024 DOWNMI0000089_MIMAT0000089 hsa-miR-31-5p 0.022548 −2.30832 DOWNMI0000735_MIMAT0004673 hsa-miR-29c-5p 0.022925 −2.30179 DOWNMI0000813_MIMAT0000762 hsa-miR-324-3p 0.023499 −2.29203 DOWNMI0000825_MIMAT0000772 hsa-miR-345-5p 0.024099 −2.28205 DOWNMI0005531_MIMAT0004909 hsa-miR-450b-5p 0.026429 −2.24524 DOWNMI0003834_MIMAT0003886 hsa-miR-769-5p 0.027019 −2.23636 DOWNMI0000808_MIMAT0000756 hsa-miR-326 0.027116 −2.23493 DOWNMI0005762_MIMAT0004983 hsa-miR-940 0.028261 −2.21824 DOWNMI0000433_MIMAT0000414 hsa-let-7g-5p 0.029329 −2.20319 DOWNMI0003589_MIMAT0003247 hsa-miR-582-5p 0.030653 −2.1852 DOWNMI0000748_MIMAT0004680 hsa-miR-130b-5p 0.030991 2.180711 UPMI0000089_MIMAT0004504 hsa-miR-31-3p 0.03322 −2.15213 DOWNMI0016902_MIMAT0019074 hsa-miR-378i 0.033939 −2.14328 DOWNMI0003190_MIMAT0002876 hsa-miR-505-3p 0.039021 −2.08488 DOWNMI0006384_MIMAT0005901 hsa-miR-1249 0.039448 −2.08028 DOWNMI0000298_MIMAT0000278 hsa-miR-221-3p 0.039583 −2.07884 DOWNMI0014197_MIMAT0015041 hsa-miR-1260b 0.039908 −2.07538 DOWNMI0014249_MIMAT0015085 hsa-miR-3200-3p 0.042866 −2.04492 DOWNMI0001448_MIMAT0001343 hsa-miR-425-3p 0.044056 −2.03317 DOWNMI0017438_MIMAT0019963 hsa-miR-4791 0.044608 −2.02781 DOWNMI0003780_MIMAT0005794 hsa-miR-1296 0.045033 −2.02372 DOWNMI0017308_MIMAT0019761 hsa-miR-4677-3p 0.045417 −2.02006 DOWNMI0000273_MIMAT0000261 hsa-miR-183-5p 0.045741 2.016984 UPMI0000283_MIMAT0000264 hsa-miR-203a 0.049459 1.983001 UP

TABLE 9 Biomarker performance in the test set AUC (95% CI) Sens^(†)Spec^(†) NPV^(†) PPV^(†) mRNA biomarker 0.66 (0.58-0.73) 0.94 0.27 0.780.60 mRNA biomarker + miR-146a-5p 0.71* (0.64-0.78)  0.93 0.33 0.83 0.59mRNA biomarker + miR-324-5p 0.65 (0.57-0.72) 0.94 0.23 0.79 0.55 mRNAbiomarker + miR-223-3p 0.67 (0.60-0.75) 0.96 0.27 0.87 0.58 mRNAbiomarker + miR-223-5p 0.67 (0.60-0.75) 0.95 0.26 0.84 0.57(*significant increase in AUC compared to mRNA biomarker p < 0.05;^(†)ROC-curve Operating Point set to 90% sensitivity in the trainingset)

human miR-146a-5p (SEQ ID NO: 2) ugagaacugaauuccauggguu human miR-324-5p(SEQ ID NO: 4) cgcauccccuagggcauuggugu human miR-223-3p (SEQ ID NO: 6)ugucaguuugucaaauacccca human miR-223-5p (SEQ ID NO: 7)cguguauuugacaagcugaguu human miR-450b-5p (SEQ ID NO: 9)uuuugcaauauguuccuga human miR-221-3p (SEQ ID NO: 11)agcuacauugucugcuggguuuc human miR-505-3p (SEQ ID NO: 13)cgucaacacuugcugguuuccu human miR-582-5p (SEQ ID NO: 15)uuacaguuguucaaccaguuacu

What is claimed herein is:
 1. A method of treating lung cancer in asubject in need thereof, the method comprising administering to thesubject an agonist of at least 1 miRNA selected from Table 10 or aninhibitor of at least 1 miRNA selected from Table 11 to the subject. 2.The method of claim 1, wherein the subject is administered an agonist ofat least 1 miRNA selected from the group consisting of: miR-146a-5p,miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p,and miR-582-5p.
 3. The method of claim 1, wherein the administering stepcomprises the administration of a vector comprising a nucleic acidencoding the agonist and/or inhibitor.
 4. The method of claim 1, whereinthe subject is a human.
 5. The method of claim 1, wherein the subject isa current or former smoker.
 6. A method of treating lung cancer in asubject in need thereof, the method comprising administering a treatmentfor lung cancer to a subject determined to have a level of expression ofat least 1 miRNAs selected from the group consisting of: miR-146a-5p,miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p,and miR-582-5p; in a sample obtained from the subject which is decreasedrelative to a reference level.
 7. The method of claim 6, furthercomprising the first steps of obtaining a sample from a subject; anddetermining the level of expression of the at least 1 miRNA; andadministering a treatment for lung cancer if the level of expression ofthe at least 1 miRNA is decreased relative to a reference level.
 8. Themethod of claim 6, wherein the level of expression is detected for atleast two miRNAs selected from the group consisting of: miR-146a-5p,miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p,and miR-582-5p.
 9. The method of claim 6, wherein the level ofexpression is detected for at least three miRNAs selected from the groupconsisting of: miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p;miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p.
 10. The method ofclaim 6, wherein the level of expression is detected for at least fourmiRNAs selected from the group consisting of: miR-146a-5p, miR-324-5p,miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, andmiR-582-5p.
 11. The method of claim 6, wherein the level of expressionis detected for at least five miRNAs selected from the group consistingof: miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,miR-221-3p-miR-505-3p, and miR-582-5p.
 12. The method of claim 6,wherein the level of expression is detected for at least six miRNAsselected from the group consisting of: miR-146a-5p, miR-324-5p,miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, andmiR-582-5p.
 13. The method of claim 6, wherein the level of expressionis detected for at least seven miRNAs selected from the group consistingof: miR-146a-5p, miR-324-5p, miR-223-3p, miR-223-5p; miR-450b-5p,miR-221-3p-miR-505-3p, and miR-582-5p.
 14. The method of claim 6,wherein the level of expression is detected for miR-146a-5p, miR-324-5p,miR-223-3p, miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, andmiR-582-5p.
 15. The method of claim 6, wherein the level of expressionis detected for at least miR-146a-5p, miR-324-5p, miR-223-3p, andmiR-223-5p.
 16. The method of claim 6, wherein the level of expressionis detected for at least miR-146a-5p.
 17. The method of claim 6, whereinthe sample is a bronchial brushing or nose epithelial sample.
 18. Themethod of claim 6, wherein the method further comprises detecting theexpression level of one or more mRNAs.
 19. The method of claim 6,wherein the expression level of no more than 100 miRNAs and/or mRNAs isdetected.
 20. The method of claim 6, wherein the treatment for lungcancer comprises administering an agonist of at least 1 miRNA selectedfrom the group consisting of: miR-146a-5p, miR-324-5p, miR-223-3p,miR-223-5p; miR-450b-5p, miR-221-3p-miR-505-3p, and miR-582-5p to thesubject.